Transcription factor modulating compounds and methods of use thereof

ABSTRACT

Substituted benzoimidazole compounds useful as anti-infectives that decrease resistance, virulence, or growth of microbes are provided. Methods of making and using substituted benzoimidazole compounds, as well as pharmaceutical preparations thereof, in, e.g., reducing antibiotic resistance and inhibiting biofilms.

RELATED APPLICATIONS

This application is is claims priority to U.S. Provisional PatentApplication 60/623,251, filed Oct. 28, 2004, U.S. Provisional PatentApplication 60/569,032, filed May 7, 2004, and U.S. Provisional PatentApplication 60/565,047, filed Apr. 23, 2004. This application is relatedto U.S. patent application Ser. No. 10/700,661, filed Nov. 3, 2003,which claims priority to U.S. Provisional Patent Application No.60/425,916, filed Nov. 13, 2002; and U.S. Provisional Patent ApplicationNo. 60/423,319, filed Nov. 1, 2002, and which is a continuation-in-partof U.S. application Ser. No. 10/139,591, filed on May 6, 2002, whichclaims priority to U.S. Provisional Patent Application Ser. No.60/288,660, entitled “Helix-Turn-Helix Protein Modulating Compounds andMethods of Use Thereof,” filed on May 4, 2001. The entire contents ofeach of the aforementioned applications are hereby incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Most antibiotics currently used and in development to treat bacterialinfections impose selective pressure on microorganisms and have led tothe development of widespread antibiotic resistance. Therefore, thedevelopment of an alternative approach to treating microbial infectionswould be of great benefit.

Multidrug resistance in bacteria is generally attributed to theacquisition of multiple transposons and plasmids bearing geneticdeterminants for different mechanisms of resistance (Gold et al. 1996.N. Engl. J. Med. 335:1445). However, descriptions of intrinsicmechanisms that confer multidrug resistance have begun to emerge. Thefirst of these was a chromosomally encoded multiple antibioticresistance (mar) locus in Escherichia coli (George and Levy, 1983. J.Bacteriol. 155:531; George and Levy 1983 J. Bacteriol. 155:541). Marmutants of E. coli arose at a frequency of 10⁻⁶ to 10⁻⁷ and wereselected by growth on subinhibitory levels of tetracycline orchloramphenicol (George and Levy, supra). These mutants exhibitedresistance to tetracyclines, chloramphenicol, penicillins,cephalosporins, puromycin, nalidixic acid, and rifampin (George andLevy, supra). Later, the resistance phenotype was extended to includefluoroquinolones (Cohen et al. 1989. Antimicrob. Agents Chemother.33:1318), oxidative stress agents (Ariza et al. 1994. J. Bacteriol.176:143; Greenberg et al. 1991. J. Bacteriol. 73:4433), and morerecently, organic solvents (White et al. 1997. J. of Bacteriology179:6122; Asako, et al. 1997. J. Bacteriol. 176:143) and householddisinfectants, e.g., pine oil and/or TRICLOSAN® (McMurry et al. 1998.FEMS Microbiology Letters 166:305; Moken et al. 1997. AntimicrobialAgents and Chemotherapy 41:2770).

The mar locus consists of two divergently positioned transcriptionalunits that flank a common promoter/operator region in E. coli,Salmonella typhimurium, and other Entrobacteriacae (Alekshun and Levy.1997, Antimicrobial Agents and Chemother. 41: 2067). One operon encodesMarC, a putative integral inner membrane protein without any yetapparent function, but which appears to contribute to the Mar phenotypein some strains. The other operon comprises marRAB, encoding the Marrepressor (MarR), which binds marO and negatively regulates expressionof marRAB (Cohen et al. 1994. J. Bacteriol. 175:1484; Martin and Rosner1995. PNAS 92:5456; Seoane and Levy. 1995 J. Bacteriol. 177:530), anactivator (MarA), which controls expression of other genes on thechromosome, e.g., the mar regulon (Cohen et al. 1994 J. Bacteriol.175:1484; Gambino et. al. 1993. J. Bacteriol. 175:2888; Seoane and Levy,1995 J. Bacteriol. 177:530), and a putative small protein (MarB) ofunknown function.

Exposure of E. coli to several chemicals, including tetracycline andchloramphenicol (Hachler et al. 1991 J Bacteriol 173(17):5532-8; Ariza,1994, J Bacteriol; 176(1): 143-8), sodium salicylate and its derivatives(Cohen, 1993, J Bacteriol; 175(24):7856-62) and oxidative stress agents(Seoane et al. 1995. J Bacteriol; 177(12):3414-9) induces the Marphenotype. Some of these chemicals act directly at the level of MarR byinteracting with the repressor and inactivating its function (Alekshun.1999. J. Bacteriol. 181:3303-3306) while others (antibiotics such astetracycline and chloramphenicol) appear to induce mar expression by analternative mechanism (Alekshun. 1999. J. Bacteriol. 181:3303-3306)e.g., through a signal transduction pathway.

Once expressed, MarA activates the transcription of several genes thatconstitute the E. coli mar regulon (Alekshun, 1997, Antimicrob. AgentsChemother. 41:2067-2075; Alekshun, 1999, J. Bacteriol. 181:3303-3306).With respect to decreased antibiotic susceptibility, the increasedexpression of the AcrAB/TolC multidrug efflux system (Fralick, 1996, JBacteriol. 178(19):5803-5; Okusu, 1996 J Bacteriol; 178(1):306-8) anddecreased synthesis of OmpF (Cohen, 1988, J Bacteriol.; 170(12):5416-22)an outer membrane protein, play major roles. Organic solvent tolerance,however, is attributed to MarA mediating increased expression of AcrAB,TolC, OmpX, and a 77 kDa protein (Aono, 1998, Extremophiles;2(3):239-48; Aono, 1998 J Bacteriol; 180(4):938-44.) but is independentof OmpF levels (Asako, 1999, Appl Environ Microbiol; 65(1):294-6).

MarA is a member of the XylS/AraC family of transcriptional activators(Gallegos et al. 1993. Nucleic Acids Res. 21:807). There are more than100 proteins within the XylS/AraC family and a defining characteristicof this group of proteins is the presence of two helix-turn-helix (HTH)DNA binding motifs. Proteins within this family activate many differentgenes, some of which produce antibiotic and oxidative stress resistanceor control microbial metabolism and virulence (Gallegos et al. supra).

SUMMARY OF THE INVENTION

The instant invention identifies microbial transcription factors, e.g.,transcription factors of the AraC-XylS family, as virulence factors inmicrobes and shows that inhibition of these factors reduces thevirulence of microbial cells. Because these transcription factorscontrol virulence, rather than essential cellular processes, thedevelopment of resistance is much less likely. Accordingly, in oneaspect, the invention is directed to a method for preventing infectionof a subject by a microbe comprising: administering a compound thatmodulates the expression or activity of a microbial transcription factorto a subject at risk of developing an infection such that infection ofthe subject is prevented.

In one embodiment, the invention pertains to a method for reducingantibiotic resistance of a microbial cell. The method includescontacting the cell with a transcription factor modulating compound ofthe formula (XI):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R¹, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R^(21a) and R^(21b) are independently selected from the group consistingof H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof; suchthat the antibiotic resistance of said cell is reduced.

In another embodiment, the invention pertains, at least in part, to amethod for reducing antibiotic resistance of a microbial cell,comprising contacting the cell with a transcription factor modulatingcompound of the formula (XII):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R²² is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, aroyl and pharmaceuticallyacceptable salts, esters and prodrugs thereof; such that the antibioticresistance of said cell is reduced.

In another embodiment, the invention includes a method for reducingantibiotic resistance of a microbial cell. The method includescontacting the cell with a transcription factor modulating compound ofthe formula (XIII):

wherein R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group, a substituted or unsubstituted straightor branched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R²³ and R²⁴ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof;

provided that when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, thenR²³ is not methyl, unsubstituted phenyl, or unsubstituted furanyl; suchthat the antibiotic resistance of said cell is reduced.

In another embodiment, the invention pertains to a method for reducingantibiotic resistance of a microbial cell, comprising contacting thecell with a transcription factor modulating compound of the formula(XIV):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R²⁵ and R²⁶ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof;

provided that when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, thenR²⁵ is not unsubstituted phenyl or O-tert-butyl; such that theantibiotic resistance of said cell is reduced.

In yet another embodiment, the invention pertains to a method forreducing antibiotic resistance of a microbial cell. The method includescontacting a cell with a transcription factor modulating compound of theformula (XV):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶ and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen;

R²⁷ is selected from the group consisting of substituted heteroaryl;substituted alkyl; substituted or unsubstituted alkenyl; alkynyl;alkylcarbonyl, arylcarbonyl; heteroarylcarbonyl; sulfonyl; alkylamino;arylamino; heteroarylamino; alkoxy, aryloxy, heteroaryloxy; substitutedstraight chain C₁-C₅ alkyl or alkenyl; substituted or unsubstitutedisoxazole, thiazolidine, imidazole, quinoline, pyrrole, triazole, orpyrazine; 2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl,and 1-fluorophenyl; and

R²⁸ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, aroyl and pharmaceuticallyacceptable salts, esters and prodrugs thereof; such that the antibioticresistance of said cell is reduced.

A method for reducing antibiotic resistance of a microbial cell,comprising contacting the cell with a transcription factor modulatingcompound of the formula (XVI):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶ and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen;

R²⁹, R³⁰ and R³¹ are independently selected from the group consisting ofH, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl, andpharmaceutically acceptable salts, esters and prodrugs thereof; suchthat the antibiotic resistance of said cell is reduced.

n yet another embodiment, the invention pertains to a method forreducing antibiotic resistance of a microbial cell. The method includescontacting the cell with a transcription factor modulating compound ofthe formula (XVII):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶ and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen;

R³² is selected from the group consisting of OH, Br, CN, CO₂H,morpholinyl, substituted aryl, substituted or unsubstituted alkenyl,alkynyl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl,arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, acyl, acylamino, alkylamino, dialkylamino,arylamino, heteroarylamino, aroyl;

R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof;

provided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is NO₂, thenR³² is not dimethylamino; and provided that when R¹ is OH, R⁴, R⁵, R⁷and R³³ are H, R⁶ is Br, then R³² is not dimethylamino; such that theantibiotic resistance of said cell is reduced.

In another embodiment, the invention pertains to a method for modulatingtranscription, by contacting a transcription factor with a transcriptionfactor modulating compound of formula (XI), (XII), (XIII), (XIV), (XV),(XVI), or (XVII).

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In one embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In another embodiment, the method further comprises administering anantibiotic.

In another aspect, the invention pertains to a method for preventingurinary tract infection of a subject by a microbe comprising:administering a compound that modulates the expression or activity of amicrobial transcription factor to a subject at risk of developing aurinary tract infection such that infection of the subject is prevented.

In yet another aspect, the invention pertains to a method for reducingvirulence of a microbe comprising: administering a compound thatmodulates the expression or activity of a microbial transcription factorto a subject at risk of developing an infection with the microbe suchthat virulence of the microbe is reduced.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In yet another embodiment, the method further comprises administering anantibiotic.

In another aspect, the invention pertains to a method for treating amicrobial infection in a subject comprising: administering a compoundthat modulates the expression or activity of a transcription factor to asubject having a microbial infection such that infection of the subjectis treated.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In still another embodiment, the invention further comprisesadministering an antibiotic.

In another aspect, the invention pertains to a method for treating aurinary tract infection in a subject comprising: administering acompound that modulates the expression or activity of a transcriptionfactor to a subject having a urinary tract infection such that infectionof the subject is treated.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In one embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In another embodiment, the method further comprises administering anantibiotic.

In another aspect, the invention pertains to a method for reducingvirulence in a microbe comprising: administering a compound thatinhibits the expression or activity of a transcription factor to asubject having a microbial infection such that virulence of the microbeis reduced.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In yet another embodiment, the method further comprises administering anantibiotic.

In another aspect, the invention pertains to a method for evaluating theeffectiveness of a compound that modulates the expression or activity ofa microbial transcription factor at inhibiting microbial virulencecomprising: infecting a non-human animal with a microbe, wherein theability of the microbe to establish an infection in the non-human animalrequires that the microbe colonize the animal; administering thecompound that modulates the expression or activity of the microbialtranscription factor to the non-human animal; and determining the levelof infection of the non-human animal, wherein the ability of thecompound to reduce the level of infection of the animal indicates thatthe compound is effective at inhibiting microbial virulence.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In yet another embodiment, the method further comprises administering anantibiotic.

In still another embodiment, the level of infection of the non-humananimal is determined by measuring the ability of the microbe to colonizethe tissue of the non-human animal.

In another embodiment, the level of infection of the non-human animal isdetermined by enumerating the number of microbes present in the tissueof the non-human animal.

In another aspect, the invention pertains to a method for identifying acompound for treating microbial infection, comprising: innoculating anon-human animal with a microbe, wherein the ability of the microbe toestablish an infection in the non-human animal requires that the microbecolonize the animal; administering a compound which reduces theexpression or activity of a microbial transcription factor to theanimal, and determining the effect of the test compound on the abilityof the microbe to colonize the animal, such that a compound for treatingmicrobial infection is identified.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In still another embodiment, the level of infection of the non-humananimal is determined by measuring the ability of the microbe to colonizethe tissue of the non-human animal.

In another embodiment, the level of infection of the non-human animal isdetermined by enumerating the number of microbes present in the tissueof the non-human animal.

In another aspect, method for identifying a compound for reducingmicrobial virulence, comprising: inoculating a non-human animal with amicrobe, wherein the ability of the microbe to establish an infection inthe non-human animal requires that the microbe colonize the animal;administering a compound which reduces the expression or activity of amicrobial transcription factor to the animal, and determining the effectof the test compound on the ability of the microbe to colonize theanimal, such that a compound for reducing microbial virulence isidentified.

In another embodiment, the transcription factor is a member of theAraC-XylS family of transcription factors.

In still another embodiment, the transcription factor is a member of theMarA family of transcription factors.

In yet another embodiment, the level of infection of the non-humananimal is determined by measuring the ability of the microbe to colonizethe tissue of the non-human animal.

In another embodiment, the level of infection of the non-human animal isdetermined by enumerating the number of microbes present in the tissueof the non-human animal.

In another aspect, the invention pertains to a method for identifyingtranscription factors which promote microbial virulence comprising:creating a microbe in which a transcription factor to be tested ismisexpressed; introducing the microbe into a non-human animal; whereinthe ability of the microbe to establish an infection in the non-humananimal requires that the microbe colonize the animal; and determiningthe ability of the microbe to colonize the animal, wherein a reducedability of the microbe to colonize the animal as compared to a wild-typemicrobial cell identifies the transcription factor as a transcriptionfactor which promotes microbial virulence.

In another embodiment, the transcription factor is a member of theAraC-XylS family of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In another embodiment, the level of infection of the non-human animal isdetermined by measuring the ability of the microbe to colonize thetissue of the non-human animal.

In another embodiment, the level of infection of the non-human animal isdetermined by enumerating the number of microbes present in the tissueof the non-human animal.

In another aspect, the invention pertains to a method for reducing theability of a microbe to adhere to an abiotic surface comprising:contacting the abiotic surface or the microbe with a compound thatmodulates the activity of a transcription factor such that the abilityof the microbe to adhere to the abiotic surface is reduced.

In one embodiment, the transcription factor is a member of the AraC-XylSfamily of transcription factors.

In another embodiment, the transcription factor is a member of the MarAfamily of transcription factors.

In yet another embodiment, the method further comprises contacting theabiotic surface or the microbe with a second agent that is effective atcontrolling the growth of the microbe.

In still another embodiment, the abiotic surface is selected from thegroup consisting of: stents, catheters, and prosthetic devices.

In one aspect, the invention pertains to a pharmaceutical compositioncomprising a compound that modulates the activity or expression of amicrobial transcription factor and a pharmaceutically acceptablecarrier, wherein the compound reduces microbial virulence.

In another aspect, the invention pertains to a pharmaceuticalcomposition comprising a compound that modulates the activity orexpression of a microbial transcription factor and an antibiotic in apharmaceutically acceptable carrier.

The present invention represents an advance over the prior art byidentifying transcription factor modulating compounds, such as, but notlimited to helix-turn-helix protein modulating compounds, and providingnovel assays that can be used to identify compounds which modulatemicrobial transcription factors, such as MarA family polypeptides andAraC family polypeptides. Modulation of gene transcription brought aboutby the modulation of transcription factors, such as helix-turn-helixdomain containing proteins, can control a wide variety of cellularprocesses. For example, in prokaryotic cells processes such asmetabolism, resistance, and virulence can be controlled.

Assays to identify compounds that are capable of modulating bacterialtranscription factors would be of great benefit in the identification ofagonists and antagonists that can be used to control gene transcriptionin both prokaryotic and eukaryotic cells.

In one embodiment, the invention pertains to a method for reducingantibiotic resistance of a cell, e.g., a eukaryotic or prokaryotic cell.In a preferred embodiment, the cell is a microbial cell. In oneembodiment, the invention pertains to a method for reducing antibioticresistance in a microbial cell, by contacting a cell with atranscription factor modulating compound, such that the antibioticresistance of the cell is reduced. In an embodiment, the transcriptionfactor modulating compound is of the formula (I):A-E  (I)wherein A is a polar moiety; E is a hydrophobic moiety, andpharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains to a method for modulatingtranscription. The method includes contacting a transcription factorwith a transcription factor modulating compound, such that thetranscription factor is modulated. The transcription factor modulatingcompound is of the formula (I):A-E  (I)wherein A is a polar moiety; and E is a hydrophobic moiety, andpharmaceutically acceptable salts thereof.

In another embodiment, the invention also includes methods foridentifying transcription factor modulating compounds. The methodincludes contacting a microbial cell with a test compound underconditions which allow interaction of the compound with the microbialcell and measuring the ability of the test compound to affect the cell.The microbial cell includes a selective marker under the direct controlof a transcription factor responsive element and a transcription factor.

In yet another embodiment, the invention includes methods foridentifying a transcription factor modulating compound. The methodincludes contacting a microbial cell comprising: 1) a selective markerunder the control of a transcription factor responsive element and 2) atranscription factor, with a test compound under conditions which allowinteraction of the compound with the microbial cell, and measuring theability of the test compound to affect the growth (e.g., in vitro or invivo) or survival of the microbial cell, wherein the inactivation of thetranscription factor leads to a decrease in in vitro or in vivo cellsurvival. The invention also pertains to similar methods where theinactivation of the transcription factor leads to an increase in cellsurvival, as well as methods wherein the activation of the transcriptionfactor leads to increased or, alternatively, decreased cell survival.

In another embodiment, the invention also pertains to methods foridentifying a transcription factor modulating compound, by contacting amicrobial cell comprising: 1) a chromosomal deletion in a guaB or purAgene, 2) heterologous guaB or purA gene under the control of itstranscription factor responsive promoter, and 3) a transcription factor,with a test compound under conditions which allow interaction of thecompound with the microbial cell. The method further includes the stepsof measuring the ability of the compound to affect gene expression ofthe reporter or the growth or survival of the microbial cell as anindication of whether the compound modulates the activity of atranscription factor. The ability of the compound to modulate theactivity of a transcription factor leads to an alteration in geneexpression may effect cell growth or survival.

The invention pertains to transcription factor modulating compounds, HTHprotein modulating compounds, and MarA family modulating compoundsidentified by the methods of the invention, methods of using thesecompounds and pharmaceutical compositions comprising these compounds.The transcription factor modulating compounds of the invention include,but are not limited to, compounds of formulae (I)-(XVII) and Tables 4and 5.

The invention also pertains to methods using computer modeling programsto identify transcription factor modulating compounds. For example, theinvention pertains to a method of identifying transcription factormodulating compounds. The method includes obtaining the structure of thetranscription factor modulating compound, and using or identifying ascaffold which has an interaction energy score of −20 or less with aportion of the transcription factor, thus identifying potentialtranscription factor modulating scaffolds.

The invention also pertains, at least in part, to a kit for identifyinga transcription factor modulating compound which modulates the activityof a transcription factor polypeptide comprising a microbial cell. Thekit includes 1) a selective marker under the control of a transcriptionfactor responsive element and 2) a transcription factor.

The invention also pertains, at least in part, to pharmaceuticalcompositions which contain an effective amount of a transcription factormodulating compound, and, optionally, a pharmaceutically acceptablecarrier.

The invention also pertains to a method of inhibiting a biofilm, byadministering a composition comprising a transcription factor modulatingcompound such that the biofilm is inhibited.

In a further embodiment, the invention pertains to a pharmaceuticalcomposition comprising an effective amount of a transcription factormodulating compound, and a pharmaceutically acceptable carrier. Thetranscription factor modulating compound is of the formula (II):

wherein

W is O or S;

X is O, S, or C, optionally linked to Q;

A¹ is C-Z⁴, O, or S;

A² is C-Z⁵, or N-Z⁵;

Z¹, Z², Z³, Z⁴ and Z⁵ are each independently hydrogen, alkoxy, hydroxy,halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, or cyano;

Z³ is hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl,heterocyclic, amino, nitro, cyano, carbonyl, or thiocarbonyl;

Q is an aromatic or heterocyclic moiety, and pharmaceutically acceptablesalts thereof.

In another further embodiment, the invention pertains to apharmaceutical composition comprising an effective amount of atranscription factor modulating compound, and a pharmaceuticallyacceptable carrier. The compound is of the formula (III):

wherein

G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl,alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;and

L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, and L¹⁰ are each independentlyoxygen, substituted or unsubstituted nitrogen, sulfur and or substitutedor unsubstituted carbon, and pharmaceutically acceptable salts thereof.

In yet another embodiment, the invention pertains to a pharmaceuticalcomposition comprising an effective amount of a transcription factormodulating compound and a pharmaceutically acceptable carrier. Thetranscription factor modulating compound is of the formula (IV):

wherein

Y¹ and Y² are each oxygen or sulfur;

J is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl,cyano, nitro, amino, or halogen;

V is substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy,alkylamino, or alkylthio;

P and K are each independently substituted or unsubstituted aryl, andpharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atranscription factor modulating compound. The transcription factormodulating compound is of the formula (V):

wherein

T¹, T², T³, T⁴, T⁵, and T⁶ are each independently substituted orunsubstituted carbon, oxygen, substituted or unsubstituted nitrogen, orsulfur;

M is hydrogen, alkyl, alkenyl, heterocyclic, alkynyl, or aryl, orpharmaceutically acceptable salts thereof.

In yet another embodiment, the invention pertains to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atranscription factor modulating compound. The transcription factormodulating compound is of the formula (Va):

wherein

R¹ is OH, OCOCO₂H, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group;

R² is H, CO₂(C₁-C₅ substituted or unsubstituted, straight or branchedalkyl), or a substituted or unsubstituted aryl group; and

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen.

In certain embodiments of formula Va, those compounds disclosed in U.S.Ser. No. 10/139,591, filed May 6, 2002, are excluded from the scope ofthe present invention.

In other embodiments of formula Va, when R⁶ is NO₂ and R² isunsubstituted phenyl, then R¹ is not O(CHCH₃)(CO₂)CH₂CH₃ or OCH₂CO₂H.Also, in another embodiment, when R⁶ is H or NO₂, then R¹ is not aphenyl-substituted alkyloxy group. In yet another embodiment, when R⁴,R⁵, R⁶, and R⁷ are all H and R² is para-methoxyphenyl, then R¹ is notOH. And in another embodiment, when R⁴, R⁵, R⁶, and R⁷ are all H and R²is unsubstituted phenyl, then R¹ is not OCH₂CO₂CH₂CH₃;

In certain aspects of formula Va, R⁴, R⁵, and R⁷ are all H.

Similarly, R¹ of formula Va may be selected from the group consisting ofOH, O(CR′R″)₁₋₃H, O(CR′R″)₁₋₃OH, O(CR′R″)₁₋₃CO₂H,O(CR′R″)₁₋₃CO₂(CR′R″)₁₋₃H, O(CR′R″)₁₋₃(CO)NH₂, O(CR′R″)₁₋₃(CNH)NH₂,OCOCO₂H, O(CR′R″)₁₋₃SO₃H, O(CR′R″)₁₋₃OSO₃H, O(CR′R″)₁₋₃PO₃H,O(CR′R″)₁₋₃OPO₃H, O(CR′R″)₁₋₃N[(CR′R″)₀₋₃H]₂, O(CR′R″)₁₋₃(CO)(NHOH), andO(CR′R″)₁₋₃(heteroaryl); wherein R′ and R″ are each independently H, aC₁-C₃ alkyl, C₂-C₃ alkenyl, or C₂-C₃ alkynyl group. Each R′ and R″ ispreferably H or CH₃.

When R¹ of formula Va is O(CR′R″)₁₋₃(heteroaryl), the heteroaryl groupmay be a pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl,imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl,pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.

Similarly, when R² of formula Va may be a substituted or unsubstitutedphenyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl,imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl,pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.

In a more particular embodiment, R⁶ of formula Va is H, (CR′R″)₁₋₃H,(CR′R″)₁₋₃OH, (CR′R″)₁₋₃NH₂, (NOH)(CR′R″)₁₋₃H, CO(CR′R″)₀₋₃NH₂,CO(CR′R″)₁₋₃H, CO(CR′R″)₁₋₃OH, CO(CR′R″)₀₋₃CF₃,(CR′R″)₀₋₃N[(CR′R″)₀₋₃H]₂, CO(substituted or unsubstituted heteroaryl),CO(C₃-C₆ substituted or unsubstituted cycloalkyl), O(CR′R″)₁₋₃H,CO(substituted or unsubstituted phenyl), CO₂(CR′R″)₀₋₃H, CN, NO₂, F, Cl,Br, or I, wherein R′ and R″ are each independently H, a C₁-C₃ alkyl,C₂-C₃ alkenyl, or C₂-C₃ alkynyl group. Preferably each R′ and R″ isindependently H or CH₃.

In yet another embodiment, R⁶ of formula Va is CO(substituted orunsubstituted heteroaryl), wherein said heteroaryl group is a pyrrolyl,furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl,tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl,pyridazinyl, or pyrimidinyl group.

In another embodiment, R⁶ of formula Va is an electron withdrawingsubstituent, selected from the group consisting of F, CF₃, NO₂,C(NOH)(CR′R″), wherein each R′ and R″ are each independently H or CH₃.

In another embodiment, the invention pertains to a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atranscription factor modulating compound. The transcription factormodulating compound may be of the formula (VI):

wherein

G¹, G², and G³ are each independently O, S, substituted or unsubstitutednitrogen, or substituted or unsubstituted carbon;

E¹, E², and E³ are each independently hydrogen, alkyl, alkenyl, alkynyl,aryl, aralkyl, or acyl; and

E⁴ is alkyl, alkenyl, alkynyl, aryl, halogen, cyano, amino, nitro, oracyl, and pharmaceutically acceptable salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a multiple sequence alignment of AraC-XylS familypolypeptides.

FIG. 2 is a multiple sequence alignment of PROSITE PS00041 and AraCfamily polypeptides.

FIG. 3 is a multiple sequence alignment of PROSITE PS01124 and AraCfamily polypeptides.

FIG. 4 is a CoMFA contour map for a representative triazinoxazepine.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention identifies microbial transcription factors, e.g.,transcription factors of the AraC-XylS family, as virulence factors inmicrobes and shows that inhibition of these factors reduces thevirulence of microbial cells. Because these transcription factorscontrol virulence, rather than essential cellular processes, modulationof these factors should not promote resistance.

Some major families of transcription factors found in bacteria includethe helix-turn-helix transcription factors (HTH) (Harrison, S. C., andA. K. Aggarwal 1990. Annual Review of Biochemistry. 59:933-969) such asAraC, MarA, Rob, SoxS and LysR; winged helix transcription factors(Gajiwala, K. S., and S. K. Burley 2000. 10:110-116), e.g., MarR,Sar/Rot family, and OmpR (Huffman, J. L., and R. G. Brennan 2002. CurrOpin Struct Biol. 12:98-106, Martinez-Hackert, E., and A. M. Stock 1997.Structure. 5:109-124); and looped-hinge helix transcription factors(Huffman, J. L., and R. G. Brennan 2002 Curr Opin Struct Biol.12:98-106), e.g. the AbrB protein family.

The AraC-XylS family of transcription factors comprises many members.MarA, SoxS, Rma, and Rob are examples of proteins within the AraC-XylSfamily of transcription factors. These factors belong to a subset of theAraC-XylS family that have historically been considered to play roles inpromoting resistance to multiple antibiotics and have not beenconsidered to be virulence factors. In fact, the role of marA invirulence has been tested using a marA null mutant of Salmonellaenterica serovar Typhimurium (S. typhimurium) in a mouse infection model(Sulavik et al. 1997. J. Bacteriology 179:1857) and no such role hasbeen found. In another model (using co-infection experiments or crudestatistics) only a weak effect of a marA null mutant in chickens hasbeen demonstrated (Randall et al. 2001. J. Med. Microbiol. 50:770). Incontrast to this earlier work, this invention is based, at least inpart, on the finding that the ability of microbes to cause infection ina host can be inhibited by inhibiting the expression and/or activity ofmicrobial transcription factors. Thus, the instant invention validatesthe use of microbial transcription factors as therapeutic targets.

The invention pertains, at least in part, to compounds which modulatetranscription factors (e.g., helix-turn-helix (HTH) proteins, AraCfamily polypeptides, MarA family polypeptides, etc.), methods ofidentifying the transcription factor modulating compounds (e.g., HTHprotein modulating compounds, AraC family polypeptide modulatingcompounds, MarA family polypeptide modulating compounds, etc.), andmethods of using the compounds.

1. Transcription Factors

The term “transcription factor” includes proteins that are involved ingene regulation in both prokaryotic and eukaryotic organisms. In oneembodiment, transcription factors can have a positive effect on geneexpression and, thus, may be referred to as an “activator” or a“transcriptional activation factor.” In another embodiment, atranscription factor can negatively effect gene expression and, thus,may be referred to as “repressors” or a “transcription repressionfactor.” Activators and repressors are generally used terms and theirfunctions are discerned by those skilled in the art.

As used herein, the term “infectivity” or “virulence” includes theability of a pathogenic microbe to colonize a host, a first steprequired in order to establish growth in a host. Infectivity orvirulence is required for a microbe to be a pathogen. In addition, avirulent microbe is one which can cause a severe infection. As usedherein, the term “pathogen” includes both obligate and opportunisticorganisms. The ability of a microbe to resist antibiotics is alsoimportant in promoting growth in a host, however, in one embodiment,antibiotic resistance is not included in the terms “infectivity” or“virulence” as used herein. Accordingly, in one embodiment, the instantinvention pertains to methods of reducing the infectivity or virulenceof a microbe without affecting (e.g., increasing or decreasing)antibiotic resistance. Preferably, as used herein, the term “infectivityor virulence” includes the ability of an organism to establish itself ina host by evading the host's barriers and immunologic defenses.

The term “AraC family polypeptide,” “AraC-XylS family polypeptide” or“AraC-XylS family peptide” include an art recognized group ofprokaryotic transcription factors which contains more than 100 differentproteins (Gallegos et al., (1997) Micro. Mol. Biol. Rev. 61: 393; Martinand Rosner, (2001) Curr. Opin. Microbiol. 4:132). AraC familypolypeptides include proteins defined in the PROSITE (PS) database(http://www.expasy.ch/prosite/) as profile PS01124. The AraC familypolypeptides also include polypeptides described in PS0041, HTH AraCFamily, 1, and PS01124, and HTH AraC Family 2. Multiple sequencealignments for the AraC-XylS family polypeptides, HTH AraC family 1, andHTH AraC family 2 are shown in FIGS. 1-3, respectively. In anembodiment, the AraC family polypeptides are generally comprised of, atthe level of primary sequence, by a conserved stretch of about 100 aminoacids, which are believed to be responsible for the DNA binding activityof this protein (Gallegos et al., (1997) Micro. Mol. Biol. Rev. 61: 393;Martin and Rosner, (2001) Curr. Opin. Microbiol. 4: 132). AraC familypolypeptides also may include two helix turn helix DNA binding motifs(Martin and Rosner, (2001) Curr. Opin. Microbiol. 4: 132; Gallegos etal., (1997) Micro. Mol. Biol. Rev. 61: 393; Kwon et al., (2000) Nat.Struct. Biol. 7: 424; Rhee et al., (1998) Proc. Natl. Acad. Sci. U.S.A.95: 10413). The term includes MarA family polypeptides and HTH proteins.In one embodiment, the invention pertains to a method for modulating anAraC family polypeptide, by contacting the AraC family polypeptide witha test compound which interacts with a portion of the polypeptideinvolved in DNA binding. In a further embodiment, the test compoundinteracts with a conserved aminoacid residue (capitalized) of the HTHAraC family 1 protein indicated in FIG. 2.

The term “helix-turn-helix protein,” “HTH protein,” “helix-turn-helixpolypeptides,” and “HTH polypeptides,” includes proteins comprising oneor more helix-turn-helix domains. Helix-turn-helix domains are known inthe art and have been implicated in DNA binding (Ann Rev. of Biochem.1984. 53:293). An example of the consensus sequence for a helix-turndomain can be found in Brunelle and Schleif (1989. J. Mol. Biol.209:607). The domain has been illustrated by the sequenceXXXPhoAlaXXPhoGlyPhoXXXXPhoXXPhoXX, where X is any amino acid and Pho isa hydrophobic amino acid.

The helix-turn-helix domain was the first DNA-binding protein motif tobe recognized. Although originally the HTH domain was identified inbacterial proteins, the HTH domain has since been found in hundreds ofDNA-binding proteins from both eukaryotes and prokaryotes. It isconstructed from two alpha helices connected by a short extended chainof amino acids, which constitutes the “turn.”

In one embodiment, a helix-turn-helix domain containing protein is a MarA family polypeptide. The language “MarA family polypeptide” includesthe many naturally occurring HTH proteins, such as transcriptionregulation proteins which have sequence similarities to MarA and whichcontain the MarA family signature pattern, which can also be referred toas an XylS/AraC signature pattern. An exemplary signature pattern whichdefines MarA family polypeptides is shown, e.g.; on PROSITE and isrepresented by the sequence:[KRQ]-[LIVMA]-X(2)-[GSTALIV]-{FYWPGDN}X(2)-[LWMSA]-X(4,9)-[LIVMF]-X(2)-[LIVMSTA]-X(2)-[GSTACIL]-X(3)-[GANQRF]-[LIVMFY]-X(4,5)-[LFY]-X(3)-[FYIVA]-{FYWHCM}-X(3)-[GSADENQKR]-X-[NSTAPKL]-[PARL],where X is any amino acid. MarA family polypeptides have two“helix-turn-helix” domains. This signature pattern was derived from theregion that follows the first, most amino terminal, helix-turn-helixdomain (HTH1) and includes the totality of the second, most carboxyterminal helix-turn-helix domain (HTH2). (See PROSITE PS00041).

The MarA family of proteins (“MarA family polypeptides”) represent onesubset of AraC-XylS family polypeptides and include proteins like MarA,SoxS, Rob, Rma, AarP, PqrA, etc. The MarA family polypeptides,generally, are involved in regulating resistance to antibiotics, organicsolvents, and oxidative stress agents (Alekshun and Levy, (1997)Antimicrob. Agents. Chemother. 41: 2067). Like other AraC-XylS familypolypeptides, MarA-like proteins also generally contain two HTH motifsas exemplified by the MarA and Rob crystal structures (Kwon et al.,(2000) Nat. Struct. Biol. 7: 424; Rhee et al., (1998) Proc. Natl. Acad.Sci. U.S.A. 95: 10413). Members of the MarA family can be identified bythose skilled in the art and will generally be represented by proteinswith homology to amino acids 30-76 and 77-106 of MarA (SEQ ID. NO. 1).

Preferably, a MarA family polypeptide or portion thereof comprises thefirst MarA family HTH domain (HTH1) (Brunelle, 1989, J Mol Biol;209(4):607-22). In another embodiment, a MarA polypeptide comprises thesecond MarA family HTH domain (HTH2) (Caswell, 1992, Biochem J.;287:493-509.). In a preferred embodiment, a MarA polypeptide comprisesboth the first and second MarA family HTH domains.

MarA family polypeptide sequences are “structurally related” to one ormore known MarA family members, preferably to MarA. This relatedness canbe shown by sequence or structural similarity between two MarA familypolypeptide sequences or between two MarA family nucleotide sequencesthat specify such polypeptides. Sequence similarity can be shown, e.g.,by optimally aligning MarA family member sequences using an alignmentprogram for purposes of comparison and comparing correspondingpositions. To determine the degree of similarity between sequences, theywill be aligned for optimal comparison purposes (e.g., gaps may beintroduced in the sequence of one protein for nucleic acid molecule foroptimal alignment with the other protein or nucleic acid molecules). Theamino acid residues or bases and corresponding amino acid positions orbases are then compared. When a position in one sequence is occupied bythe same amino acid residue or by the same base as the correspondingposition in the other sequence, then the molecules are identical at thatposition. If amino acid residues are not identical, they may be similar.As used herein, an amino acid residue is “similar” to another amino acidresidue if the two amino acid residues are members of the same family ofresidues having similar side chains. Families of amino acid residueshaving similar side chains have been defined in the art (see, forexample, Altschul et al. 1990. J. Mol. Biol. 215:403) including basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan). The degree (percentage) ofsimilarity between sequences, therefore, is a function of the number ofidentical or similar positions shared by two sequences (i.e., %homology=# of identical or similar positions/total # of positions×100).Alignment strategies are well known in the art; see, for example,Altschul et al. supra for optimal sequence alignment.

MarA family polypeptides may share some amino acid sequence similaritywith MarA. The nucleic acid and amino acid sequences of MarA as well asother MarA family polypeptides are available in the art. For example,the nucleic acid and amino acid sequence of MarA can be found, e.g., onGeneBank (accession number M96235 or in Cohen et al. 1993. J. Bacteriol.175:1484, or in SEQ ID NO:1 and SEQ ID NO:2.

The nucleic acid and/or amino acid sequences of MarA can be used as“query sequences” to perform a search against databases (e.g., eitherpublic or private) to, for example, identify other MarA family membershaving related sequences. Such searches can be performed, e.g., usingthe NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990)J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performedwith the NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to MarA family nucleic acid molecules. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to MarA proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

MarA family members can also be identified as being similar based ontheir ability to specifically hybridize to nucleic acid sequencesspecifying MarA. Such stringent conditions are known to those skilled inthe art and can be found e.g., in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,non-limiting example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.Conditions for hybridizations are largely dependent on the meltingtemperature Tm that is observed for half of the molecules of asubstantially pure population of a double-stranded nucleic acid. Tm isthe temperature in ° C. at which half the molecules of a given sequenceare melted or single-stranded. For nucleic acids of sequence 11 to 23bases, the Tm can be estimated in degrees C. as 2(number of A+Tresidues)+4(number of C+G residues). Hybridization or annealing ofnucleic acid molecules should be conducted at a temperature lower thanthe Tm, e.g., 15° C., 20° C., 25° C. or 30° C. lower than the Tm. Theeffect of salt concentration (in M of NaCl) can also be calculated, seefor example, Brown, A., “Hybridization” pp. 503-506, in The Encyclopediaof Molec. Biol., J. Kendrew, Ed., Blackwell, Oxford (1994).

Preferably, the nucleic acid sequence of a MarA family member identifiedin this way is at least about 10%, 20%, more preferably at least about30%, more preferably at least about 40% identical and preferably atleast about 50%, or 60% identical to a MarA nucleotide sequence. Inpreferred embodiments, the nucleic acid sequence of a MarA family memberis at least about 70%, 80%, preferably at least about 90%, morepreferably at least about 95% identical with a MarA nucleotide sequence.Preferably, MarA family members have an amino acid sequence at leastabout 20%, preferably at least about 30%, more preferably at least about40% identical and preferably at least about 50%, or 60% or moreidentical with a MarA amino acid sequence. In preferred embodiments, thenucleic acid sequence of a MarA family member is at least about 70%,80%, more preferably at least about 90%, or more preferably at leastabout 95% identical with a MarA nucleotide sequence. However, it will beunderstood that the level of sequence similarity among microbialregulators of gene transcription, even though members of the samefamily, is not necessarily high. This is particularly true in the caseof divergent genomes where the level of sequence identity may be low,e.g., less than 20% (e.g., B. burgdorferi as compared e.g., to B.subtilis). Accordingly, structural similarity among MarA family memberscan also be determined based on “three-dimensional correspondence” ofamino acid residues. As used herein, the language “three-dimensionalcorrespondence” is meant to includes residues which spatiallycorrespond, e.g., are in the same position of a MarA family polypeptidemember as determined, e.g., by x-ray crystallography, but which may notcorrespond when aligned using a linear alignment program. The language“three-dimensional correspondence” also includes residues which performthe same function, e.g., bind to DNA or bind the same cofactor, asdetermined, e.g., by mutational analysis.

Exemplary MarA family polypeptides are shown in Table 1, FIGS. 1-3, andat Prosite (PS00041) and include: AarP, Ada, AdaA, AdiY, AfrR, AggR,AppY, AraC, CafR, CelD, CfaD, CsvR, D90812, EnvY, ExsA, FapR, HrpB, InF,InvF, LcrF, LumQ, MarA, MelR, MixE, MmsR, MsmR, OrfR, Orf_f375, PchR,PerA, PocR, PqrA, RafR, RamA, RhaR, RhaS, Rns, Rob, SoxS, S52856, TetD,TcpN, ThcR, TmbS, U73857, U34257, U21191, UreR, VirF, XylR, XylS, Xys1,2, 3, 4, Ya52, YbbB, YfiF, YisR, YzbC, and YijO. The nucleotide andamino acid sequences of the E. coli Rob molecule are shown in SEQ IDNO:3 and 4, respectively. TABLE 1 Some Bacterial MarA homologs^(a)Gram-negative bacteria Escherichia coli MarA (1) OrfR (2, 3) SoxS (4, 5)AfrR (6) AraC (7) CelD (8) D90812 (9) FapR (10, 11) MelR (12) ORF f375(13, 14) RhaR (15, 16, 17) RhaS (18) Rob (19) U73857 (20) XylR (21) YijO(22) Proteus vulgaris PqrA (23) Salmonella typhimurium MarA (24) InvF(25) PocR (26) Kiebsiella pneumoniae RamA (27) Haemophilus influenzaeYa52 (28) Yersinia spp. CafR (29) LcrF (30) or VirF (30) Providenciastuartii AarP (31) Pseudomonas spp. MmsR (32) TmbS (33) XylS (34) Xys1,2, 3, 4 (35, 36) Cyanobacteria Synechocystis spp. LumQ (37) PchR (37)Gram-positive bacteria Lactobacillus helveticus U34257 (38) Azorhizobiumcaulinodans S52856 (39) Streptomyces spp. U21191 (40) AraL (41)Streptococcus mutans MsmR (42) Pediococcus pentosaceus RafR (43)Photobacterium leiognathi LumQ (44) Bacillus subtilis AdaA (45) YbbB(46) YfiF (47) YisR (48) YzbC (49)^(a)The smaller MarA homologs, ranging in size from 87 (U34257) to 138(OrfR) amino acid residues, are represented in boldface. References aregiven in parentheses and are listed below.References for Table 1:(1) S. P. Cohen, et al. 1993. J. Bacteriol. 175: 1484-1492(2) G. M. Braus, et al. 1984. J. Bacteriol. 160: 504-509(3) K. Schollmeier, et al., 1984. J. Bacteriol. 160: 499-503(4) C. F. Amabile-Cuevas, et al., 1991. Nucleic Acids Res. 19: 4479-4484(5) J. Wu, et al., 1991. J. Bacteriol. 173: 2864-2871(6) M. K. Wolf, et al., 1990. Infect. Immun. 58: 1124-1128(7) C. M. Stoner, et al.. 1982. J. Mol Biol. 153: 649-652(8) L. L. Parker, et al., 1990. Genetics 123: 455-471(9) H. Mori, 1996. Unpublished data taken from the NCBI databases(10) P. Klaasen, et al., 1990. Mol. Microbiol. 4: 1779-1783(11) M. Ahmed, et al., 1994. J. Biol. Chem 269-28506-28513(12) C. Webster, et al., 1989. Gene 83: 207-213(13) G. Plunkett, III. 1995. Unpublished(14) C Garcia-Martin, et al., 1992. J. Gen. Microbiol. 138: 1109-1116(15) G. Plunkett, III., et al. 1993. Nucleic Acids Res. 21: 3391-3398(16) C. G. Tate, et al. 1992. J. Biol. Chem. 267: 6923-6932(17) J. F. Tobin et al., 1987. J. Mol. Biol. 196: 789-799(18) J. Nishitani, 1991. Gene 105: 37-42(19) R. E. Benz, et al., 1993. Zentralbl. Bakteriol. Parasitenkd.Infektionskr. Hyg. Abt. 1 Orig. 278: 187-196(20) M. Duncan, et al., 1996. Unpublished data(21) H. J. Sofia, et al, 1994. Nucleic Acids Res. 22: 2576-2586(22) F. R. Blattner, et al., 1993. Nucleic Acids Res. 21: 5408-5417(23) H. Ishida, et al., 1995. Antimicrob. Agents Chemother. 39: 453-457(24) M. C. Sulavik, et al., 1997. J. Bacteriol. 179: 1857-1866(25) K. Kaniga, et al., 1994. Mol. Microbiol. 13: 555-568(26) J. R. Roth, et al. 1993. J. Bacteriol. 175: 3303-3316(27) A. M. George, et al., 1983. J. Bacteriol. 155: 541-548(28) R. D. Fleischmann, et al., 1995. Science 269: 469-512(29) E. E. Galyov, et al., 1991. FEBS Lett. 286: 79-82(30) N. P. Hoe, et al., 1992. J. Bacteriol. 174: 4275-4286(31) G. Cornelis, et al., 1989. J. Bacteriol. 171: 254-262(32) D. R. Macinga, et al., 1995. J. Bacteriol. 177: 3407-3413(33) M. I. Steele, et al., 1992. J. Biol. Chem. 267: 13585-13592(34) G. Deho, et al., 1995. Unpublished data(35) N. Mermod, et al., 1984. EMBO J. 3: 2461-2466(36) S. J. Assinder, et al., 1992. Nucleic Acids Res. 20: 5476(37) S. J. Assinder, et al., 1993. J. Gen. Microbiol. 139: 557-568(38) E. G. Dudley, et al., 1996. J. Bacteriol. 178: 701-704(39) D. Geelen, et al., 1995. Unpublished data(40) J. Kormanec, et al., 1995. Gene 165: 77-80(41) C. W. Chen, et al., 1992. J. Bacteriol. 174: 7762-7769(42) R. R. Russell, et al., 1992. J. Biol. Chem, 267: 4631-4637(43) K. K. Leenhouts, et al., 1995. Unpublished data(44) J. W. Lin, et al., 1995. Biochem. Biophys. Res. Commun. 217:684-695(45) F. Morohoshi, et al. 1990. Nucleic Acids Res. 18: 5473-5480(46) M. Rosenberg, et al., 1979. Annu. Rev. Genet. 13: 319-353(47) H. Yamamoto, et al., 1996. Microbiology 142: 1417-1421(48) L. B. Bussey, et al., 1993. J. Bacteriol. 175: 6348-6353(49) P. G. Quirk, et al., 1994. Biochim. Biophys. Acta 1186: 27-34

The term “transcription factor modulating compound” or transcriptionfactor modulator” includes HTH protein modulating compounds, HTH proteinmodulators. Transcription factor modulating compounds include compoundswhich interact with one or more transcription factors, such that theactivity of the transcription factor is modulated, e.g., enhanced orinhibited. The term also includes both AraC family modulating compoundsand MarA family modulating compounds. In one embodiment, thetranscription factor modulating compound is an inhibiting compound of atranscription factor, e.g., a prokaryotic transcription factor or aeukaryotic transcription activation factor. In one embodiment, thetranscription factor modulating compounds modulate the activity of atranscription factor as measured by assays known in the art or LANCEassays such as those described in Example 8. In one embodiment, thetranscription factor modulating compound inhibits a particulartranscription factor by about 10% or greater, about 40% or greater,about 50% or greater, about 60% or greater, about 70% or greater, about80% or greater, about 90% or greater, about 95% or greater, or about100% as compared to the activity of the transcription factor with outthe transcription factor modulating compound. In another embodiment, thetranscription factor modulating compound inhibits biofilm formation. Inone embodiment, the transcription factor modulating compound inhibitsbiofilm formation as measured by assays known in the art or the CrystalViolet assay described in Example 7. In one embodiment, thetranscription factor of the invention inhibits the formation of abiofilm by about 25% or more, 50% or more, 75% or more, 80% or more, 90%or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% ormore, 99.9% or more, 99.99% or more, or by 100%, as compared to theformation of a biofilm without the transcription factor modulatingcompound.

The term “HTH protein modulating compound” or “HTH protein modulator”includes compounds which interact with one or more HTH proteins suchthat the activity of the HTH protein is modulated, e.g., enhanced or,inhibited. In one embodiment, the HTH protein modulating compound is aMarA family polypeptide modulating compound. In one embodiment, theactivity of the HTH protein is enhanced when it interacts with the HTHprotein modulating compound. For example, the activity of the HTHprotein may be increased by greater than 10%, greater the 20%, greaterthan 50%, greater than 75%, greater than 80%, greater than 90%, or 100%of the activity of the HTH protein in the absence of the HTH modulatingcompound. In another embodiment, the activity of the HTH protein isdecreased upon an interaction with the HTH protein modulating compound.In an embodiment, the activity of the HTH protein is decreased by about25% or more, 50% or more, 75% or more, 80% or more, 90% or more, 95% ormore, 96% or more, 97% or more, 98% or more, 99% or more, 99.9% or more,99.99% or more, or by 100%, as compared to the activity of the proteinof a HTH protein when not contacted with an HTH modulating compound ofthe invention using techniques and assays described herein. Values andranges included and/or intermediate of the values set forth herein arealso intended to be within the scope of the present invention.

The term “MarA family polypeptide modulating compound” or “MarA familymodulating compound” include compounds which interact with one or moreMarA family polypeptides such that the activity of the MarA familypeptide is enhanced or inhibited. In an embodiment, the MarA familypolypeptide modulating compound is an inhibiting compound. In a furtherembodiment, the MarA family inhibiting compound is an inhibitor of MarA,Rob, and/or SoxS. In another embodiment, the MarA family polypeptidemodulating compound modulates the expression of luciferase in theLuciferase Assay described in Example 9. In one embodiment, the MarAfamily polypeptide modulating compound decreases luciferase expressionby greater than 10%, greater than 20%, greater than 30%, greater than40%, greater than 50%, greater than 60%, greater than 70%, greater than80%, greater than 90% or about 100%.

The term “polypeptide(s)” refers to a peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” includes both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, Proteins—Structure And Molecular Properties, 2^(nd) Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in Posttranslational Covalent Modification Of Proteins, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

As used herein, the term “winged helix” includes dimeric transcriptionfactors in which each monomer comprises a helix-turn-helix motiffollowed by one or two β-hairpin wings (Brennan. 1993. Cell. 74:773;Gajiwala and Burley. 2000. Curr. Opin. Struct. Biol. 10:110). Theclassic winged helix motif comprises two wings, three a helices, andthree p strands in the sequence H1-B1-H2-T-H3-B2-W1-B3-W2 (where H is ahelix, B is a β strand, T is a turn, and W is a wing), although somevariation in structure has been demonstrated (Huffman and Brennan. 2002.Current Opinion in Structural Biology. 12:98).

As used herein the term “looped-hinge helix” included transcriptionfactors, such as AbrB which, in the absence of DNA, have revealed adimeric N-terminal region consisting of a four-stranded P sheet and aC-terminal DNA-binding region comprising one a helix and a “loopedhinge” (see, e.g., Huffman and Brennan. 2002 Current Opinion inStructural Biology 12:98). Residues corresponding to R23 and R24 of AbrBare critical for DNA recognition and contribute to the electropositivenature of the DNA-binding region.

Preferred polypeptides (and the nucleic acid molecules that encode them)are “naturally occurring.” As used herein, a “naturally-occurring”molecule refers to a molecule having an amino acid or a nucleotidesequence that occurs in nature (e.g., a natural polypeptide). Inaddition, naturally or non-naturally occurring variants of thepolypeptides and nucleic acid molecules which retain the same functionalactivity, (such as, the ability to bind to target nucleic acid molecules(e.g., comprising a marbox) or to polypeptides (e.g. RNA polymerase)with a naturally occurring polypeptide are provided for. Suchimmunologic cross-reactivity can be demonstrated, e.g., by the abilityof a variant to bind to a MarA family polypeptide responsive element.Such variants can be made, e.g., by mutation using techniques that areknown in the art. Alternatively, variants can be chemically synthesized.

As used herein the term “variant(s)” includes nucleic acid molecules orpolypeptides that differ in sequence from a reference nucleic acidmolecule or polypeptide, but retain its essential properties. Changes inthe nucleotide sequence of the variant may, or may not, alter the aminoacid sequence of a polypeptide encoded by the reference nucleic acidmolecule. Nucleotide or amino acid changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by a naturally occurring reference sequence. Atypical variant of a polypeptide differs in amino acid sequence from areference polypeptide. Generally, differences are limited so that thesequences of the reference polypeptide and the variant are closelysimilar overall and, in many regions, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, additions, and/or deletions in any combination.

A variant of a nucleic acid molecule or polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally. Non-naturally occurring variants ofnucleic acid molecules and polypeptides may be made from a referencenucleic acid molecule or polypeptide by mutagenesis techniques, bydirect synthesis, and by other recombinant methods known to skilledartisans. Alternatively, variants can be chemically synthesized. Forinstance, artificial or mutant forms of autologous polypeptides whichare functionally equivalent, (e.g., have the ability to interact with aMarA family polypeptide responsive element) can be made using techniqueswhich are well known in the art.

Mutations can include, e.g., at least one discrete point mutation whichcan give rise to a substitution, or by at least one deletion orinsertion. For example, mutations can also be made by random mutagenesisor using cassette mutagenesis. For the former, the entire coding regionof a molecule is mutagenized by one of several methods (chemical, PCR,doped oligonucleotide synthesis) and that collection of randomly mutatedmolecules is subjected to selection or screening procedures. In thelatter, discrete regions of a polypeptide, corresponding either todefined structural or functional determinants are subjected tosaturating or semi-random mutagenesis and these mutagenized cassettesare re-introduced into the context of the otherwise wild type allele. Inone embodiment, PCR mutagenesis can be used. For example, Megaprimer PCRcan be used (O. H. Landt, 1990. Gene 96:125-128).

In preferred embodiments, a MarA family polypeptide excludes one or moreof XylS, AraC, and MelR. In other preferred embodiments, a MarA familypolypeptide is involved in antibiotic resistance. In particularlypreferred embodiments, a MarA family polypeptide is selected from thegroup consisting of: MarA, RamA, AarP, Rob, SoxS, and PqrA.

The language “activity of a transcription factor” includes the abilityof a transcription factor to interact with DNA, e.g., to bind to atranscription factor responsive promoter, or to initiate transcriptionfrom such a promoter. The language expressly includes the activities ofAraC family polypeptides, HTH proteins and MarA family polypeptides.

The language “activity of a MarA family polypeptide” includes theability of the MarA family polypeptide to interact with DNA, e.g., tobind to a MarA family polypeptide responsive promoter, or to initiatetranscription from such a promoter. MarA functions both as atranscriptional activator (e.g., upregulating genes such as inaA, galT,micF, etc.) and as a repressor (e.g., downregulating genes such as fecA,purA, guaB, etc.) (Alekshun, 1997, Antimicrob. Agents Chemother.41:2067-2075; Barbosa & Levy, J. Bact. 2000, Vol. 182, p. 3467-3474;Pomposiello et al. J. Bact. 2001, Vol 183, p. 3890-3902).

The language “transcription factor responsive element” includes anucleic acid sequence which can interact with a transcription factor(e.g., promoters or enhancers or operators) which are involved ininitiating transcription of an operon in a microbe. Transcription factorresponsive elements responsive to various transcription factors areknown in the art and additional responsive elements can be identified byone of ordinary skill in the art. For example, microarray analysis canbe used to identify genes that are regulated by a transcription factorof interest. For interest, genes regulated by a transcription factorwould be expressed at higher levels in wild type cells than in cellswhich are deleted for the transcription factor. In addition, genesresponsive to a given transcription factor would comprise one or moretarget sequences responsive to the transcription factor in theirpromoter regions (Lyons et al. 2000. PNAS 97:7957). Exemplary responsiveelements include: araBAD, araE, araFGH (responsive to AraC); melBAD(responsive to MelR); rhaSR (responsive to RhaR); rahBAD, rhaT(responsive to RhaS); Pm (responsive to XylS); fumC, inaA, micF, nfo,pai5, sodA, tolC, acrAB, fldA, fpr, mar, poxB, ribA, and zwf (responsiveto MarA, SoxS, Rob); and coo, ms (responsive to Rns).

The language “marA family polypeptide responsive element” includes anucleic acid sequence which can interact with marA, e.g., promoters orenhancers which are involved in regulating transcription of a nucleicacid sequence in a microbe. MarA responsive elements compriseapproximately 16 base pair marbox sequence, the sequence critical forthe binding of MarA to its target. In addition, a secondary site, theaccessory marbox, upstream of the primary marbox contributes to basaland derepressed mar transcription. A marbox may be situated in eitherthe forward or backward orientation. (Martin, 1999, Mol. Microbiol.34:431-441). In the marRAB operon, the marbox is in the backwardorientation and is thus located on the sense strand with respect tomarRAB (Martin, 1999, Mol. Microbiol. 34:431-441). Subtle differenceswithin the marbox sequence of particular promoters may account fordifferential regulation by MarA and other related, e.g., SoxS and Rob,transcription factors (Martin, 2000, Mol Microbiol; 35(3):623-34). Inone embodiment, MarA family responsive elements are promoters that arestructurally or functionally related to a marA promoter, e.g., interactwith MarA or a protein related to MarA. Preferably, the marA familypolypeptide responsive element is a marRAB promoter. For example, in themar operon, several promoters are marA family polypeptide responsivepromoters as defined herein, e.g., the 405-bp ThaI fragment from themarO region is a marA family responsive promoter (Cohen et al. 1993. J.Bact. 175:7856). In addition, MarA has been shown to bind to a 16 bpMarA binding site (referred to as the “marbox” within marO (Martin etal. 1996. J. Bacteriol. 178:2216). MarA also affects transcription fromthe acrAB; micF; mlr 1, 2, 3; slp; nfo; inaA; fpr; sodA; soi-17, 19;zwf; fumC; or rpsF promoters (Alekshun and Levy. 1997. AntimicrobialAgents and Chemother. 41:2067). Other marA family responsive promotersare known in the art and include: araBAD, araE, araFGH and araC, whichare activated by AraC; Pm, which is activated by XylS; melAB which isactivated by MelR; and oriC which is bound by Rob.

The language “MarA family polypeptide responsive promoter” also includesportions of the above promoters which are sufficient to activatetranscription upon interaction with a MarA family member protein. Theportions of any of the MarA family polypeptide-responsive promoterswhich are minimally required for their activity can be easily determinedby one of ordinary skill in the art, e.g., using mutagenesis. Exemplarytechniques are described by Gallegos et al. (1996, J. Bacteriol.178:6427). A “MarA family polypeptide responsive promoter” also includesnon-naturally occurring variants of MarA family polypeptide responsivepromoters which have the same function as naturally occurring MarAfamily promoters. Preferably such variants have at least 30% or greater,40% or greater, or 50% or greater, nucleotide sequence identity with anaturally occurring MarA family polypeptide responsive promoter. Inpreferred embodiments, such variants have at least about 70% nucleotidesequence identity with a naturally occurring MarA family polypeptideresponsive promoter. In more preferred embodiments, such variants haveat least about 80% nucleotide sequence identity with a naturallyoccurring MarA family polypeptide responsive promoter. In particularlypreferred embodiments, such variants have at least about 90% nucleotidesequence identity and preferably at least about 95% nucleotide sequenceidentity with a naturally occurring MarA family polypeptide responsivepromoter. In yet other embodiments nucleic acid molecules encodingvariants of MarA family polypeptide responsive promoters are capable ofhybridizing under stringent conditions to nucleic acid moleculesencoding naturally occurring MarA family polypeptide responsivepromoters.

In one embodiment, the methods described herein can employ moleculesidentified as responding to the transcription factors of the invention,i.e., molecules in a regulon whose expression is controlled by thetranscription factor. For example, compounds that modulate transcriptionof genes that are directly modulated by a microbial transcription factor(e.g., a marA family transcription factor) can be used to modulatevirulence of a microbe or modulate infection by a microbe. In anotherembodiment, such genes can be identified as important in controllingvirulence using the methods described herein. As used herein, the term“regulon” includes two or more loci in two or more different operonswhose expression is regulated by a common repressor or activatorprotein.

The term “interact” includes close contact between molecules thatresults in a measurable effect, e.g., the binding of one molecule withanother. For example, a MarA family polypeptide can interact with a MarAfamily polypeptide responsive element and alter the level oftranscription of DNA. Likewise, compounds can interact with a MarAfamily polypeptide and alter the activity of a MarA family polypeptide.

The term “inducible promoter” includes promoters that are activated toinduce the synthesis of the genes they control. As used herein, the term“constitutive promoter” includes promoters that do not require thepresence of an inducer, e.g., are continuously active.

The terms “heterologous DNA” or “heterologous nucleic acid” includes DNAthat does not occur naturally in the cell (e.g., as part of the genome)in which it is present or which is found in a location or locations inthe genome that differ from that in which it occurs in nature or whichis operatively linked to DNA to which it is not normally linked innature (i.e., a gene that has been operatively linked to a heterologouspromoter). Heterologous DNA is 1) not naturally occurring in aparticular position (e.g., at a particular position in the genome) or 2)is not endogenous to the cell into which it is introduced, but has beenobtained from another cell. Heterologous DNA can be from the samespecies or from a different species. Any DNA that one of skill in theart would recognize or consider as heterologous or foreign to the cellin which is expressed is herein encompassed by the term heterologousDNA.

The terms “heterologous protein”, “recombinant protein”, and “exogenousprotein” are used interchangeably throughout the specification and referto a polypeptide which is produced by recombinant DNA techniques,wherein generally, DNA encoding the polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. That is, the polypeptide isexpressed from a heterologous nucleic acid molecule.

The term “microbe” includes microorganisms expressing or made to expressa transcription factor, araC family polypeptide, HTH protein, or a marAfamily polypeptide. “Microbes” are of some economic importance, e.g.,are environmentally important or are important as human pathogens. Forexample, in one embodiment microbes cause environmental problems, e.g.,fouling or spoilage, or perform useful functions such as breakdown ofplant matter. In another embodiment, microbes are organisms that live inor on mammals and are medically important. Preferably microbes areunicellular and include bacteria, fungi, or protozoa. In anotherembodiment, microbes suitable for use in the invention aremulticellular, e.g., parasites or fungi. In preferred embodiments,microbes are pathogenic for humans, animals, or plants. Microbes may beused as intact cells or as sources of materials for cell-free assays. Inone embodiment, the microbes include prokaryotic organisms. In otherembodiments, the microbes include eukaryotic organisms. Exemplarybacteria that comprise MarA homologs include the following: MarA E. coliUPEC (uropathogenic) EPEC (enteropathogenic) Salmonella entericaCholerasuis (septicemia) Enteritidis enteritis Typhimurium enteritisTyphimurium (multi-drug resistant) Yersinia enterocolitica Yersiniapestis Pseudomonas aeruginosa Enterobacter spp. Klebsiella sp. Proteusspp. Vibrio cholerae Shigella sp. Providencia stuartii Neisseriameningitidis Mycobacterium tuberculosis Mycobacterium lepraeStaphylococcus aureus Streptococcus pyogenes Enterococcus faecalisBordetella pertussis Bordetella bronchiseptica

The term selective marker includes polypeptides that serve asindicators, e.g., provide a selectable or screenable trait whenexpressed by a cell. The term “selective marker” includes bothselectable markers and counterselectable markers. As used herein theterm “selectable marker” includes markers that result in a growthadvantage when a compound or molecule that fulfills the test parameterof the assay is present. The term “counterselectable marker” includesmarkers that result in a growth disadvantage unless a compound ormolecule is present which disrupts a condition giving rise to expressionof the counterselectable marker. Exemplary selective markers includecytotoxic gene products, gene products that confer antibioticresistance, gene products that are essential for growth, gene productsthat confer a selective growth disadvantage when expressed in thepresence of a particular metabolic substrate (e.g., the expression ofthe URA3 gene confers a growth disadvantage in the presence of5-fluoroorotic acid).

As used herein the term “reporter gene” includes any gene which encodesan easily detectable product which is operably linked to a regulatorysequence, e.g., to a transcription factor responsive promoter. Byoperably linked it is meant that under appropriate conditions an RNApolymerase may bind to the promoter of the regulatory region and proceedto transcribe the nucleotide sequence such that the reporter gene istranscribed. In preferred embodiments, a reporter gene consists of thetranscription factor responsive promoter linked in frame to the reportergene. In certain embodiments, however, it may be desirable to includeother sequences, e.g, transcriptional regulatory sequences, in thereporter gene construct. For example, modulation of the activity of thepromoter may be effected by altering the RNA polymerase binding to thepromoter region, or, alternatively, by interfering with initiation oftranscription or elongation of the mRNA. Thus, sequences which areherein collectively referred to as transcriptional regulatory elementsor sequences may also be included in the reporter gene construct. Inaddition, the construct may include sequences of nucleotides that altertranslation of the resulting mRNA, thereby altering the amount ofreporter gene product.

Examples of reporter genes include, but are not limited to CAT(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature282: 864-869) luciferase, and other enzyme detection systems, such asbeta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell.Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984),PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667);PhoA, alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182:231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placentalsecreted alkaline phosphatase (Cullen and Malim (1992) Methods inEnzymol. 216:362-368) and green fluorescent protein (U.S. Pat. No.5,491,084; WO96/23898).

In certain embodiments of the invention it will be desirable to obtain“isolated or recombinant” nucleic acid molecules transcription factorsor mutant forms thereof. The term “isolated or recombinant” includesnucleic acid molecules which have been, e.g., (1) amplified in vitro by,for example, polymerase chain reaction (PCR); (2) recombinantly producedby cloning, or (3) purified, as by cleavage and gel separation; or (4)synthesized by, for example, chemical synthesis. Such a nucleic acidmolecule is isolated from the sequences which naturally flank it in thegenome and from cellular components.

In yet other embodiments of the invention, it will be desirable toobtain a substantially purified or recombinant transcription factor.Such polypeptides, for example, can be purified from cells which havebeen engineered to express an isolated or recombinant nucleic acidmolecule which encodes a transcription factor. For example, as describedin more detail below, a bacterial cell can be transformed with a plasmidwhich encodes a transcription factor. The transcription factor can thenbe purified from the bacterial cells and used, for example, in thecell-free assays described herein or known in the art.

As used herein, the term “antibiotic” includes antimicrobial agentsisolated from natural sources or chemically synthesized. The term“antibiotic” refers to antimicrobial agents for use in human therapy.Preferred antibiotics include: tetracycline, fluoroquinolones,chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid,and rifampin.

The term “test compound” includes any reagent or test agent which isemployed in the assays of the invention and assayed for its ability toinfluence the activity of a transcription factor, e.g., an AraC familypolypeptide, an HTH protein, or a MarA family polypeptide, e.g., bybinding to the polypeptide or to a molecule with which it interacts.More than one compound, e.g., a plurality of compounds, can be tested atthe same time for their ability to modulate the activity of atranscription factor, e.g., an AraC family polypeptide, an HTH protein,or a MarA family polypeptide, activity in a screening assay. In anadvantageous embodiment, the test compound is a MarA family modulatingcompound.

Test compounds that can be tested in the subject assays includeantibiotic and non-antibiotic compounds. In one embodiment, testcompounds include candidate detergent or disinfectant compounds.Exemplary test compounds which can be screened for activity include, butare not limited to, peptides, non-peptidic compounds, nucleic acids,carbohydrates, small organic molecules (e.g., polyketides), and naturalproduct extract libraries. The term “non-peptidic test compound”includes compounds that are comprised, at least in part, of molecularstructures different from naturally-occurring L-amino acid residueslinked by natural peptide bonds. However, “non-peptidic test compounds”also include compounds composed, in whole or in part, of peptidomimeticstructures, such as D-amino acids, non-naturally-occurring L-aminoacids, modified peptide backbones and the like, as well as compoundsthat are composed, in whole or in part, of molecular structuresunrelated to naturally-occurring L-amino acid residues linked by naturalpeptide bonds. “Non-peptidic test compounds” also are intended toinclude natural products.

In one embodiment, small molecules can be used as test compounds. Theterm “small molecule” is a term of the art and includes molecules thatare less than about 1000 molecular weight or less than about 500molecular weight. In one embodiment, small molecules do not exclusivelycomprise peptide bonds. In another embodiment, small molecules are notoligomeric. Exemplary small molecule compounds which can be screened foractivity include, but are not limited to, peptides, peptidomimetics,nucleic acids, carbohydrates, small organic molecules (e.g.,polyketides) (Cane et al. 1998. Science 282:63), and natural productextract libraries. In another embodiment, the compounds are small,organic non-peptidic compounds. In a further embodiment, a smallmolecule is not biosynthetic.

The term “antagonist” includes transcription factor modulating compounds(e.g., AraC family polypeptide modulating compounds, HTH proteinmodulating compounds, MarA family polypeptide modulating compounds,etc.) which inhibit the activity of a transcription factor by binding toand inactivating the transcription factor (e.g., an AraC familymodulating compound, an MarA family polypeptide modulating compound,etc.), by binding to a nucleic acid target with which the transcriptionfactor interacts (e.g., for MarA, a marbox), by disrupting a signaltransduction pathway responsible for activation of a particular regulon(e.g., for Mar, the inactivation of MarR or activation of MarAsynthesis), and/or by disrupting a critical protein-protein interaction(e.g., MarA-RNA polymerase interactions that are required for MarA tofunction as a transcription factor.) Antagonists may include, forexample, naturally or chemically synthesized compounds such as smallcell permeable organic molecules, nucleic acid interchelators, peptides,etc.

The term “agonist” includes transcription factor modulating compounds(e.g., AraC family polypeptide modulating compounds, HTH proteinmodulating compounds, MarA family polypeptide modulating compounds,etc.) which promote the activity of a transcription factor by binding toand activating the transcription factor (e.g., an AraC family modulatingcompound, an MarA family polypeptide modulating compound, etc.), bybinding to a nucleic acid target with which the transcription factorinteracts (e.g., for MarA, a marbox), by facilitating a signaltransduction pathway responsible for activation of a particular regulon(e.g., for Mar, the inactivation of MarR or activation of MarAsynthesis), and/or by facilitating a critical protein-proteininteraction (e.g., MarA-RNA polymerase interactions that are requiredfor MarA to function as a transcription factor.) Agonists may include,for example, naturally or chemically synthesized compounds such as smallcell permeable organic molecules, nucleic acid interchelators, peptides,etc.

II. MarA Family Polypeptide Helix-Turn-Helix Domains

Helix-turn-helix domains are known in the art and have been implicatedin DNA binding (Ann Rev. of Biochem. 1984. 53:293). An example of theconsensus sequence for a helix-turn domain can be found in Brunelle andSchleif (1989, J. Mol. Biol. 209:607). The domain has been illustratedby the sequence XXXPhoAlaXXPhoGlyPhoXXXXPhoXXPhoXX, where X is any aminoacid and Pho is a hydrophobic amino acid.

The crystal structure of MarA has been determined and the first (mostamino terminal) HTH domain of MarA has been identified as comprisingfrom about amino acid 31 to about amino acid 52 and the second HTHdomain of MarA has been identified as comprising from about amino acid79 to about amino acid 102 (Rhee et al. 1998. Proc. Natl. Acad. Sci.USA. 95:10413).

Locations of the helix-turn-helix domains in other MarA family membersas well as other HTH proteins can easily be found by one of skill in theart. For example using the MarA protein sequence and an alignmentprogram, e.g., the ProDom program or other programs known in the art, aportion of the MarA amino acid sequence e.g., comprising one or both HTHdomains of MarA (such as from about amino acid 30 to about amino acid107 of MarA) to produce an alignment. Using such an alignment, the aminoacid sequences corresponding to the HTH domains of MarA can beidentified in other MarA family member proteins. An exemplary consensussequence for the first helix-turn-helix domain of a MarA familypolypeptide can be illustrated as XXXXAXXXXXSXXXLXXXFX, where X is anyamino acid. An exemplary consensus sequence for the secondhelix-turn-helix domain of a MarA family polypeptide is illustrated asXXIXXIAXXXGFXSXXXFXXX[F/Y], where X is any amino acid. Preferably, aMarA family polypeptide first helix-turn-helix domain comprises theconsensus sequence E/D-X-V/L-A-D/E-X-A/S-G-X-S-X3-L-Q-X2-F-K/R/E-X2-T/I.Preferably, a MarA family polypeptide second helix-turn-helix domaincomprises the consensus sequence I-X-D-1-A-X3-G-F-X-S-X2-F-X3-F-X4.

In an embodiment, a MarA family member HTH domain is a MarA HTH 10domain. The first and second helix-turn-helix domains of MarA are,respectively, EKVSERSGYSKWHLQRMFKKET and ILYLAERYGFESQQTLTRTFKNYF. Otherexemplary MarA family helix-turn-helix domains include: about amino acid210 to about amino acid 229 and about amino acid 259 to about amino acid278 of MelR; about amino acid 196 to about amino acid 215 and aboutamino acid 245 to about amino acid 264 of AraC; and about amino acid 230to about amino acid 249 (or 233-253) and about amino acid 281 to aboutamino acid 301 (or 282-302) of XylS (see e.g., Brunelle et al. 1989. J.Mol. Biol. 209:607; Niland et al. 1996. J. Mol. Biol. 264:667; Gallegoset al. 1997. Microbiology and Molecular Biology Reviews. 61:393).

“MarA family polypeptide helix-turn-helix domains” are derived from orare homologous to the helix-turn-helix domains found in the MarA familypolypeptides as described supra. In preferred embodiments, a MarA familypolypeptide excludes one or more of XylS, AraC, and MelR. Inparticularly preferred embodiments, a MarA family polypeptide isselected from the group consisting of: MarA, RamA, AarP, Rob, SoxS, andPqrA.

Both of the helix-turn-helix domains present in MarA family polypeptidesare in the carboxy terminal end of the protein. Proteins or portionsthereof comprising either or both of these domains can be used in theinstant methods. In certain embodiments, a polypeptide which is used inscreening for compounds comprises the helix-turn-helix domain mostproximal to the carboxy terminus (HTH2) of the MarA family polypeptidefrom which it is derived. In other embodiments, such a polypeptidecomprises the helix-turn-helix domain most proximal to the aminoterminus (HTH1) of the MarA family polypeptide from which it is derived.In one embodiment, other polypeptide sequences may also be present,e.g., sequences that might facilitate immobilizing the domain on asupport, or, alternatively, might facilitate the purification of thedomain.

In an embodiment, such a polypeptide consists essentially of thehelix-turn-helix domain most proximal to the carboxy terminus of theMarA family polypeptide from which it is derived. In other preferredembodiments, such a polypeptide consists essentially of thehelix-turn-helix domain most proximal to the amino terminus of the MarAfamily polypeptide from which it is derived.

In an embodiment, such a polypeptide consists of the helix-turn-helixdomain most proximal to the carboxy terminus of the AraC familypolypeptide or MarA family polypeptide from which it is derived. Inother preferred embodiments, such a polypeptide consists of thehelix-turn-helix domain most proximal to the amino terminus of the AraCfamily polypeptide or MarA family polypeptide from which it is derived.

MarA family polypeptide or AraC family polypeptide helix-turn-helixdomains can be made using techniques which are known in the art. Thenucleic acid and amino acid sequences of transcription factors, such asMarA family polypeptides, are available, for example, from GenBank.Using this information, the helix-turn-helix consensus motif andmutational analysis provided herein, one of ordinary skill in the artcan identify MarA family or AraC family polypeptide helix-turn-helixdomains.

In certain embodiments of the invention it will be desirable to obtain“isolated or recombinant” nucleic acid molecules encoding transcriptionfactors or portions thereof (e.g., HTH protein helix-turn-helix domains,AraC family helix-turn-helix domains, MarA family helix-turn-helixdomains or mutant forms thereof). By “isolated or recombinant” is meanta nucleic acid molecule which has been (1) amplified in vitro by, forexample, polymerase chain reaction (PCR); (2) recombinantly produced bycloning, or (3) purified, as by cleavage and gel separation; or (4)synthesized by, for example, chemical synthesis. Such a nucleic acidmolecule is isolated from the sequences which naturally flank it in thegenome and from cellular components.

The isolated or recombinant nucleic acid molecules encodingtranscription factors (e.g., HTH protein helix-turn-helix domains, AraCfamily helix-turn-helix domains, MarA family helix-turn-helix domains ormutant forms thereof) can then, for example, be utilized in bindingassays, can be expressed in a cell, or can be expressed on the surfaceof phage, as discussed further below.

In yet other embodiments of the invention, it will be desirable toobtain a substantially purified or recombinant HTH proteinhelix-turn-helix domains (e.g., MarA family helix-turn-helix domains ormutant forms thereof). Such polypeptides, for example, can be purifiedfrom cells which have been engineered to express an isolated orrecombinant nucleic acid molecule which encodes a HTH proteinhelix-turn-helix domain (e.g., MarA family helix-turn-helix domain ormutant forms thereof). For example, as described in more detail below, abacterial cell can be transformed with a plasmid which encodes a MarAfamily helix-turn-helix domain. The MarA family helix-turn-helix proteincan then be purified from the bacterial cells and used, for example, inthe cell-free assays described herein.

Purification of a HTH protein helix-turn-helix domain (e.g., MarA familyhelix-turn-helix domain) can be accomplished using techniques known inthe art. For example, column chromatography could be used, or antibodiesspecific for the domain or for a polypeptide fused to the domain can beemployed, for example on a column or in a panning assay.

In preferred embodiments, cells used to express HTH proteinhelix-turn-helix domains (e.g., MarA family helix-turn-helix domains ormutant forms thereof) for purification, e.g., host cells, comprise amutation which renders any endogenous HTH proteins nonfunctional orcauses the endogenous protein to not be expressed. In other embodiments,mutations may also be made in MarR or related genes of the host cell,such that repressor proteins which bind to the same promoter as a MarAfamily polypeptide are not expressed by the host cell.

In certain embodiments of the invention, it will be desirable to use amutant form of a HTH protein helix-turn helix domain, e.g., anon-naturally occurring form of a MarA family helix-turn-helix domainwhich has altered activity, e.g., does not retain wild type MarA familypolypeptide helix-turn-helix domain activity, or which has reducedactivity or which is more active when compared to a wild-type MarAfamily polypeptide helix-turn-helix domain.

Such mutant forms can be made using techniques which are well known inthe art. For example, random mutagenesis can be used. Using randommutagenesis one can mutagenize an entire molecule or one can proceed bycassette mutagenesis. In the former instance, the entire coding regionof a molecule is mutagenized by one of several methods (chemical, PCR,doped oligonucleotide synthesis) and that collection of randomly mutatedmolecules is subjected to selection or screening procedures. In thesecond approach, discrete regions of a protein, corresponding either todefined structural or functional determinants (e.g., the first or secondalpha helix of a helix-turn-helix domain) are subjected to saturating orsemi-random mutagenesis and these mutagenized cassettes arere-introduced into the context of the otherwise wild type allele.

In a preferred embodiment, PCR mutagenesis is used. For example, Example2 describes the use of Megaprimer PCR(O. H. Landt, Gene 96:125-128) usedto introduce an NheI restriction site into the centers of both the helixA (position 1989) and helix B (position 2016) regions of the marA gene.

In one embodiment, such mutant helix-turn-helix domains comprise one ormore mutations in the helix-turn-helix domain most proximal to thecarboxy terminus (HTH2) of the MarA family polypeptide molecule. In apreferred embodiment, the mutation comprises an insertion into helix Aand helix B of the helix-turn-helix domain most proximal to the carboxyterminus of the MarA family polypeptide. In one embodiment, such mutanthelix-turn-helix domains comprise one or more mutations in thehelix-turn-helix domain most proximal to the amino terminus (HTH1) ofthe MarA family polypeptide molecule. In a preferred embodiment, themutation comprises an insertion into helix A and helix B of thehelix-turn-helix domain most proximal to the amino terminus of the MarAfamily polypeptide. In particularly preferred embodiments, the mutationis selected from the group consisting of: an insertion at an amino acidcorresponding to about position 33 of MarA and an insertion at an aminoacid position corresponding to about position 42 of MarA.“Corresponding” amino acids can be determined, e.g., using an alignmentof the helix-turn-helix domains.

Such mutant forms of MarA family helix-turn-helix motifs are useful ascontrols to verify the specificity of antiinfective compounds for a MarAfamily helix-turn-helix domain or as controls for the identification ofgenetic loci which affect resistance to antiinfectives. For example, themutant MarA family helix-turn-helix domains described in the appendedExamples demonstrate that insertional inactivation of MarA at eitherhelix A or helix B in the first HTH domain abolished the multidrugresistance phenotype in both E. coli and M. smegmatis. By the use of anassay system such as that described in Example 2, which demonstrates theability of MarA family polypeptide helix-turn-helix domains to increaseantibiotic resistance and that mutant forms of these domains do not havethe same effect, one can clearly show that the response of any geneticloci identified is specific to a MarA family helix-turn-helix domain.

II. Expression of Polypeptide or Portions Thereof

Nucleic acids encoding transcription factors, such as AraC familypolypeptides, HTH proteins, e.g., MarA family polypeptides or selectablemarkers (or portions thereof that retain an activity of the full-lengthpolypeptide, e.g., are capable of binding to a transcription factorresponsive element or retain their indicator function) can be expressedin cells using vectors. Almost any conventional delivery vector can beused. Such vectors are widely available commercially and it is withinthe knowledge and discretion of one of ordinary skill in the art tochoose a vector which is appropriate for use with a given microbialcell. The sequences encoding these domains can be introduced into a cellon a self-replicating vector or may be introduced into the chromosome ofa microbe using homologous recombination or by an insertion element suchas a transposon.

These nucleic acids can be introduced into microbial cells usingstandard techniques, for example, by transformation using calciumchloride or electroporation. Such techniques for the introduction of DNAinto microbes are well known in the art. In one embodiment, a nucleicacid molecule which has been amplified in vitro by, for example,polymerase chain reaction (PCR); recombinantly produced by cloning, or)purified, as by cleavage and gel separation; or synthesized by, forexample, chemical synthesis can be used to produce MarA familypolypeptides (George, A. M. & Levy, S. B. (1983) J. Bacteriol. 155,541-548; Cohen, S. P. et al. (1993) J. Infect. Dis. 168, 484-488; Cohen,S. P et al. (1993) J. Bacteriol. 175, 1484-1492; Sulavick, M. C. et al.(1997) J. Bacteriol. 179, 1857-1866).

Host cells can be genetically engineered to incorporate nucleic acidmolecules of the invention. In one embodiment nucleic acid moleculesspecifying transcription factors can be placed in a vector. The term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid molecule to which it has been linked. The term“expression vector” or “expression system” includes any vector, (e.g., aplasmid, cosmid or phage chromosome) containing a gene construct in aform suitable for expression by a cell (e.g., linked to a promoter). Inthe present specification, “plasmid” and “vector” are usedinterchangeably, as a plasmid is a commonly used form of vector.Moreover, the invention is intended to include other vectors which serveequivalent functions. A great variety of expression systems can be usedto produce the polypeptides of the invention. Such vectors include,among others, chromosomal, episomal and virus-derived vectors, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids.

Appropriate vectors are widely available commercially and it is withinthe knowledge and discretion of one of ordinary skill in the art tochoose a vector which is appropriate for use with a given host cell. Thesequences encoding a transcription factor, such as, for example, MarAfamily polypeptides, can be introduced into a cell on a self-replicatingvector or may be introduced into the chromosome of a microbe usinghomologous recombination or by an insertion element such as atransposon.

The expression system constructs may contain control regions thatregulate expression. “Transcriptional regulatory sequence” is a genericterm to refer to DNA sequences, such as initiation signals, enhancers,operators, and promoters, which induce or control transcription ofpolypeptide coding sequences with which they are operably linked. Itwill also be understood that a recombinant gene encoding a transcriptionfactor gene, e.g., an HTH protein gene or an AraC family polypeptide,e.g., MarA family polypeptide, can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring transcription factor gene. Exemplary regulatorysequences are described in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Forinstance, any of a wide variety of expression control sequences, thatcontrol the expression of a DNA sequence when operatively linked to it,may be used in these vectors to express DNA sequences encoding thepolypeptide.

Generally, any system or vector suitable to maintain, propagate orexpress nucleic acid molecules and/or to express a polypeptide in a hostmay be used for expression in this regard. The appropriate DNA sequencemay be inserted into the expression system by any of a variety ofwell-known and routine techniques, such as, for example, those set forthin Sambrook et al., Molecular Cloning, A Laboratory Manual, (supra).

Exemplary expression vectors for expression of a gene encoding apolypeptide and capable of replication in a bacterium, e.g., a grampositive, gram negative, or in a cell of a simple eukaryotic fungus suchas a Saccharomyces or, Pichia, or in a cell of a eukaryotic organismsuch as an insect, a bird, a mammal, or a plant, are known in the art.Such vectors may carry functional replication-specifying sequences(replicons) both for a host for expression, for example a Streptomyces,and for a host, for example, E. coli, for genetic manipulations andvector construction. See, e.g., U.S. Pat. No. 4,745,056. Suitablevectors for a variety of organisms are described in Ausubel, F. et al.,Short Protocols in Molecular Biology, Wiley, New York (1995), and forexample, for Pichia, can be obtained from Invitrogen (Carlsbad, Calif.).

Useful expression control sequences, include, for example, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat polypeptide, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. A useful translational enhancer sequenceis described in U.S. Pat. No. 4,820,639.

In one embodiment, an inducible promoter will be employed to express apolypeptide of the invention. For example, in one embodiment, trp(induced by tryptophan), tac (induced by lactose), or tet (induced bytetracycline) can be used in bacterial cells, or GAL1 (induced bygalactose) can be used in yeast cell.

In another embodiment, a constitutive promoter can be used to express apolypeptide of the invention.

It should be understood that the design of the expression vector maydepend on such factors as the choice of the host cell to be transformedand/or the type of polypeptide desired to be expressed. Representativeexamples of appropriate hosts include bacterial cells, such as grampositive, gram negative cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoplera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

In one embodiment, cells used to express heterologous polypeptides ofthe invention, comprise a mutation which renders one or more endogenoustranscription factors, such as a AraC family polypeptide or a MarAfamily polypeptide, nonfunctional or causes one or more endogenouspolypeptide to not be expressed. Manipulation of the genetic backgroundin this manner allows for screening for compounds that modulate specifictranscription factors, such as MarA family members or AraC familymembers, or more than one transcription factors.

In other embodiments, mutations may also be made in other related genesof the host cell, such that there will be no interference from theendogenous host loci. In yet another embodiment, a mutation may be madein a chromosomal gene to create a heterotroph.

Introduction of a nucleic acid molecule into the host cell(“transformation”) can be effected by methods described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology, (1986) and Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989). Examples include calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

Purification of polypeptides, e.g., recombinantly expressedpolypeptides, can be accomplished using techniques known in the art. Forexample, if the polypeptide is expressed in a form that is secreted fromcells, the medium can be collected. Alternatively, if the polypeptide isexpressed in a form that is retained by cells, the host cells can belysed to release the polypeptide. Such spent medium or cell lysate canbe used to concentrate and purify the polypeptide. For example, themedium or lysate can be passed over a column, e.g., a column to whichantibodies specific for the polypeptide have been bound. Alternatively,such antibodies can be specific for a second polypeptide which has beenfused to the first polypeptide (e.g., as a tag) to facilitatepurification of the first polypeptide. Other means of purifyingpolypeptides are known in the art.

IV. Methods for Identifying Antiinfective Compounds which Modulate anActivity of a Transcription Factor

Transcription factor agonists and antagonists can be assayed in avariety of ways. For example, in one embodiment, the invention providesfor methods for identifying a compound which modulates an transcriptionfactor, e.g., by measuring the ability of the compound to interact withan transcription factor nucleic acid molecule or an transcription factorpolypeptide or the ability of a compound to modulate the activity orexpression of an transcription factor polypeptide. Furthermore, theability of a compound to modulate the binding of an transcription factorpolypeptide or transcription factor nucleic acid molecule to a moleculeto which they normally bind, e.g., a nucleic acid or protein moleculecan be tested.

In one embodiment, a transcription factor and its cognate DNA sequencecan be present in a cell free system, e.g., a cell lysate and the effectof the compound on that interaction can be measured using techniquesknown in the art.

In a preferred embodiment, the assay system is a cell-based system.Compounds identified using the subject methods are useful, e.g., tointerfere with the ability of a microbe to grow in a host and/or inreducing microbial virulence and, thereby, and in reducing the abilityof the microbe to cause infection in a host.

The ability of the test compound to modulate the expression and/oractivity of a transcription factor can be determined in a variety ofways. Exemplary methods which can be used in the instant assays areknown in the art and are described, e.g., in U.S. Pat. No. 5,817,793 andWO 99/61579. Other exemplary methods are described in more detail below.

In one embodiment, the invention provides for methods of identifying atest compound which modulates the expression and/or activity of atranscription factor, (e.g., an HTH protein, a MarA family polypeptide,an AraC family polypeptide, etc.) by contacting a cell expressing atranscription factor (or portion thereof) with a test compound underconditions which allow interaction of the test compound with the cell.

Assays

In one embodiment, the expression of a selectable marker that confers aselective growth disadvantage or lethality is placed under the directcontrol of a MarA responsive element in a cell expressing marA.

In one embodiment, marA is plasmid encoded. In one embodiment, thegenetic background of the host organism is manipulated, e.g., to deleteone or more chromosomal marA genes or marA homolog genes.

In one embodiment, expression of marA is controlled by a highlyregulated and inducible promoter. For example, in one embodiment, apromoter selected from the group consisting of trp, tac, or tet inbacterial cells or GAL1 in yeast cells can be used.

In another embodiment, expression of marA is constitutive.

In one embodiment, a selective marker is a cytotoxic gene product (e.g.,ccdB).

In another embodiment, a selective marker is a gene that confersantibiotic resistance (e.g., kan, cat, or bla).

In another embodiment, a selective marker is an essential gene (e.g.,purA or guaB in a purine or guanine heterotroph).

In still another embodiment, a selective marker is a gene that confers aselective growth disadvantage in the presence of a particular metabolicsubstrate (e.g., the expression of URA3 in the presence of5-fluoroorotic acid [5-FOA] in yeast).

In one embodiment, compounds that modulate transcription factors (e.g.,HTH proteins, AraC family polypeptides, or MarA family polypeptides) areidentified using a one-hybrid screening assay. As used herein, the term“one-hybrid screen” as used herein includes assays that detect thedisruption of protein-nucleic acid interactions. These assays willidentify agents that interfere with the binding of a transcriptionfactor (e.g., an HTH protein, a AraC family polypeptide, or a MarAfamily polypeptide) to a particular target, e.g., DNA containing, forMarA, a marbox, at the level of the target itself, e.g., by binding tothe target and preventing the trnscriptional activation factor frominteracting with or binding to this site.

In another embodiment, compounds of the invention are identified using atwo-hybrid screening assay. As used herein the term “two-hybrid screen”as used herein includes assays that detect the disruption ofprotein-protein interactions. Such two hybrid assays can be used tointerfere with crucial protein-transcription factor interactions (e.g.,HTH protein interactions, AraC family polypeptide interactions, MarAfamily polypeptide interactions). One example would be to prevent RNApolymerase-MarA family polypeptide contacts, that are necessary for theMarA family polypeptide to function as a transcription factor (eitherpositive acting or negative acting).

In yet another embodiment, compounds of the invention are identifiedusing a three-hybrid screening assay. As used herein the term“three-hybrid screen” as used herein includes assays that will detectthe disruption of a signal transduction pathway(s) required for theactivation of a particular regulon of interest. In one embodiment, thethree-hybrid screen is used to detect disruption of a signaltransduction pathway(s) required for the activation of the Mar regulon,i.e., synthesis of MarA. (Li and Park. J. Bact. 181:4824). The assay canbe used to identify compounds that may be responsible for activatingtranscription factor expression, e.g., Mar induction by antibiotics mayproceed in this manner.

In one embodiment of the assay, the expression of a selective marker(e.g., ccdB, cat, bla, kan, guaB, URA3) is put under the direct controlof an activatable MarA responsive activatable promoter (e.g., inaA,galT, micF). In the absence of Mar A, the expression of the selectivemarker would be silent. For example, in the case of regulation of thecytotoxic gene ccdB, the gene would be silent and the cells wouldsurvive. Synthesis of MarA from an inducible plasmid in a suitable hostwould result in the activation of the MarA responsive activatablepromoter and expression of the selective marker. In the case of ccdB,the gene would be expressed and result in cell death. Compounds thatinhibit MarA would be identified as those that permit cell survivalunder conditions of MarA expression.

In another embodiment, e.g., where the expression of the MarA responsiveactivatable promoter regulates a gene such as URA3, a different resultcould be obtained. In this case, in the absence of MarA and thus, in theabsence of URA3 expression, cells would grow in the presence of a 5-FOA.Upon activation of MarA expression and thus synthesis of URA3, cellswould die following the conversion of 5-FOA to a toxic metabolite byURA3.

In another embodiment, a selectable marker is put under the directcontrol of a repressible MarA responsive promoter (e.g., fecA). In thisexample, under conditions of constitutive MarA synthesis, e.g., in aconstitutive mar (marc) mutant the expression of the selectable markerwould be silent. In the case of ccdB, this would mean that cells wouldremain viable. Following inactivation of MarA, the selectable markerwould be turned on, resulting in cell death.

In another embodiment, a purine or guanine heterotroph can beconstructed by the inactivation of the chromosomal guaB or purA genes inE. coli. The guaB or purA gene would then be cloned into a suitablevector, under the control of its natural promoter. This construct wouldthen be transformed into the heterotrophic host. The heterotroph willnot grow if MarA expression is constitutive and if cells are grown onmedia lacking purines or guanine. This can be attributed to MarAmediated repression of guaB or purA synthesis. Candidate inhibitingcompounds of MarA can be identified as compounds that restored growth,i.e., relieved MarA mediated repression of guaB and purA expression. Inanother embodiment, genes that are required for growth in vivo, forexample in an animal model of infection.

In preferred embodiments, controls may be included to ensure that anycompounds which are identified using the subject assays do not merelyappear to modulate the activity of a transcription factor, because theyinhibit protein synthesis. For example, if a compound appears to inhibitthe synthesis of a protein being translated from RNA which istranscribed upon activation of a MarA family responsive element, it maybe desirable to show that the synthesis of a control, e.g., a proteinwhich is being translated from RNA which is not transcribed uponactivation of a MarA family responsive element, is not affected by theaddition of the same compound. For example, the amount of the MarAfamily polypeptide being made and compared to the amount of anendogenous protein being made. In another embodiment the microbe couldbe transformed with another plasmid comprising a promoter which is not aMarA family responsive promoter and a protein operably linked to thatpromoter. The expression of the control protein could be used tonormalize the amount of protein produced in the presence and absence ofcompound.

V. Microbes Suitable for Testing

Numerous different microbes are suitable for testing in the instantassays. As such, they may be used as intact cells or as sources ofmaterial, e.g., nucleic acid molecules or polypeptides as describedherein.

In preferred embodiments, microbes for use in the claimed methods arebacteria, either Gram negative or Gram positive bacteria. Morespecifically, any bacteria that are shown to become resistant toantibiotics, e.g., to display a Mar phenotype are preferred for use inthe claimed methods, or that are infectious or potentially infectious.

Examples of microbes suitable for testing include, but are not limitedto, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonasacidovorans, Pseudomonas alcaligenes, Pseudomonas putida,Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonashydrophilia, Escherichia coli, Citrobacter freundii, Salmonellatyphimurium, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae,Klebsiella oxytoca, Serratia marcescens, Francisella tularensis,Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providenciaalcalifaciens, Providencia rettgeri, Providencia stuartii, Acinetobactercalcoaceticus, Acinetobacter haemolyticus, Yersinia enterocolitica,Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia,Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae,Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilusducreyi, Pasteurella multocida, Pasteurella haemolytica, Branhamellacatarrhalis, Helicobacter pylori, Campylobacter fetus, Campylobacterjejuni, Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae,Yibrio parahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Gardnerella vaginalis,Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homologygroup, Bacteroides vulgatus, Bacteroides ovalus, Bacteroidesthetaiotaomicron, Bacteroides uniformis, Bacteroides eggerthii,Bacteroides splanchnicus, Clostridium difficile, Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium intracellulare,Mycobacterium leprae, Corynebacterium diphtheriae, Corynebacteriumulcerans, Streptococcus pneumoniae, Streptococcus agalactiae,Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Staphylococcus intermedius, Staphylococcus hyicus subsp.hyicus, Staphylococcus haemolyticus, Staphylococcus hominis, andStaphylococcus saccharolyticus.

In one embodiment, microbes suitable for testing are bacteria from thefamily Enterobacteriaceae. In preferred embodiments, the compound iseffective against a bacteria of a genus selected from the groupconsisting of: Escherichia, Proteus, Salmonella, Klebsiella,Providencia, Enterobacter, Burkholderia, Pseudomonas, Aeromonas,Haemophilus, Yersinia, Neisseria, and Mycobacteria.

In yet other embodiments, the microbes to be tested are Gram positivebacteria and are from a genus selected from the group consisting of:Lactobacillus, Azorhizobium, Streptomyces, Pediococcus, Photobacterium,Bacillus, Enterococcus, Staphylococcus, Clostridium, and Streptococcus.

In other embodiments, the microbes to be tested are fungi. In apreferred embodiment the fungus is from the genus Mucor or Candida,e.g., Mucor racmeosus or Candida albicans.

In yet other embodiments, the microbes to be tested are protozoa. In apreferred embodiment the microbe is a malaria or cryptosporidiumparasite.

VI. Transcription Factor Modulating Compounds and Test Compounds

Compounds for testing in the instant methods can be derived from avariety of different sources and can be known or can be novel. In oneembodiment, libraries of compounds are tested in the instant methods toidentify transcriptional activation factor modulating compounds, e.g.,HTH protein modulating compounds, AraC family polypeptide modulatingcompounds, MarA family polypeptide modulating compounds, etc. In anotherembodiment, known compounds are tested in the instant methods toidentify transcription factor modulating compounds (such as, forexample, HTH protein modulating compounds, AraC family polypeptidemodulating compounds, MarA family polypeptide modulating compounds,etc.). In an embodiment, compounds among the list of compounds generallyregarded as safe (GRAS) by the Environmental Protection Agency aretested in the instant methods. In another embodiment, the transcriptionfactors which are modulated by the modulating compounds are ofprokaryotic microbes.

A recent trend in medicinal chemistry includes the production ofmixtures of compounds, referred to as libraries. While the use oflibraries of peptides is well established in the art, new techniqueshave been developed which have allowed the production of mixtures ofother compounds, such as benzodiazepines (Bunin et al. 1992. J. Am.Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678)oligocarbamates (Cho et al. 1993. Science. 261:1303), and hydantoins(DeWitt et al. supra). Rebek et al. have described an approach for thesynthesis of molecular libraries of small organic molecules with adiversity of 104-105 (Carell et al. 1994. Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. Angew. Chem. Int. Ed. Engl. 1994. 33:2061).

The compounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries, synthetic library methods requiringdeconvolution, the ‘one-bead one-compound’ library method, and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. Anticancer Drug Des. 1997.12:145).

Exemplary compounds which can be screened for activity include, but arenot limited to, peptides, nucleic acids, carbohydrates, small organicmolecules, and natural product extract libraries. In one embodiment, thetest compound is a peptide or peptidomimetic. In another, preferredembodiment, the compounds are small, organic non-peptidic compounds.

Other exemplary methods for the synthesis of molecular libraries can befound in the art, for example in: Erb et al. 1994. Proc. Natl. Acad.Sci. USA 91:11422; Horwell et al. 1996 Immunopharmacology 33:68; and inGallop et al. 1994. J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.). Other types of peptide libraries may also be expressed, see,for example, U.S. Pat. Nos. 5,270,181 and 5,292,646). In still anotherembodiment, combinatorial polypeptides can be produced from a cDNAlibrary.

In other embodiments, the compounds can be nucleic acid molecules. Inpreferred embodiments, nucleic acid molecules for testing are smalloligonucleotides. Such oligonucleotides can be randomly generatedlibraries of oligonucleotides or can be specifically designed to reducethe activity of a transcription factor, e.g., a HTH protein, a MarAfamily polypeptide, or an AraC family polypeptide. For example, in oneembodiment, these oligonucleotides are sense or antisenseoligonucleotides. In an embodiments, oligonucleotides for testing aresense to the binding site of a particular transcription factor, e.g., aMarA family polypeptide helix-turn-helix domain. Methods of designingsuch oligonucleotides given the sequences of a particular transcriptionfactor polypeptide, such as a MarA family polypeptide, is within theskill of the art.

In yet another embodiment, computer programs can be used to identifyindividual compounds or classes of compounds with an increasedlikelihood of modulating a transcription factor activity, e.g., an HTHprotein, a AraC family polypeptide, or a MarA family polypeptideactivity. Such programs can screen for compounds with the propermolecular and chemical complementarities with a chosen transcriptionfactor. In this manner, the efficiency of screening for transcriptionfactor modulating compounds in the assays described above can beenhanced.

VII. Computer Modeling Techniques for Identifying Transcription FactorModulating Compounds

The invention also pertains to the use of molecular design techniques todesign transcription factor modulating compounds, e.g., HTH proteinmodulating compounds, AraC family modulating compounds, MarA familymodulating compounds, or MarA modulating compounds, which are capable ofbinding or interacting with one or more transcription factors (e.g., ofa prokaryotic or eukaryotic organism). The invention pertains to boththe transcription factor modulating compounds identified by the methodsas well as the modeling methods, and compositions comprising thecompounds identified by the methods.

In an embodiment, the invention pertains to a method of identifyingtranscription factor modulating compounds. The method includes obtainingthe structure of a transcription factor of interest, and using GLIDE toidentify a scaffold which has an interaction energy score of −20 or less(e.g., −40 or less, e.g., −60 or less) with a portion of thetranscription factor.

The invention pertains, at least in part, to a computational screeningof small molecule databases for chemical entities or compounds that canbind in whole, or in part, to a transcription factor, such as a HTHprotein, an AraC family polypeptide, a MarA family polypeptide, e.g.,MarA. In this screening, the quality of fit of such entities orcompounds to the binding site may be judged either by shapecomplementarity or by estimated interaction energy (Meng, E. C. et al.,1992, J. Coma. Chem., 13:505-524). Such a procedure allows for thescreening of a very large library of potential transcription factormodulating compounds for the proper molecular and chemicalcomplementarities with a selected protein or class or proteins.Transcription factor modulating compounds identified throughcomputational screening can later be passed through the in vivo assaysdescribed herein as further screens. For example, a MarA inhibitingcompound identified through computational screening could be tested forits ability to promote cell survival in a cell system containing acounterselectable marker under the control a MarA activated promoter.The promotion of cell survival in the foregoing assay would beindicative of a compound that inhibits MarA's activity as atranscriptional activator. Other suitable assays are described in theExamples and through the specification.

The crystal structures of both MarA (PDB ID code 1BL0) and its homologRob (PDB ID code 1DY5) are available in the Protein Data Bank(http://www.rcsb.org/pdb/). These structures were used to identify siteson the proteins that could be targeted by small molecule chemicalinhibiting compounds. A total of at least eight potential small moleculebinding sites on MarA (Table 2) and four sites on Rob (Table 3) wereidentified as potential small molecule binding sites. The inventionpertains, at least in part, to MarA modulating compounds which interactwith any one of the following sites of MarA (based on the sequence givenin SEQ ID NO. 2). TABLE 2 Site Number Residues (based on full lengthMarA) Site Label 1 42 to 50 R46 Major Groove 2 54 to 62 L56 HTH core 355 to 65 R61 Minor Groove 4 15 to 25 W19 5 14 to 25 E21 6 24 to 35 L28 776 to 83 P78 8 106 to 112 R110

The GLIDE docking method was then used to fit combinatorial chemistryscaffolds into these sites and an interaction energy was calculated foreach. Eight scaffolds were predicted to bind to site 1, encompassingamino acids tryptophan 42 to lysine 50, with an interaction energy scoreof −60 or less. These scaffolds are shown below:

Three scaffolds were identified for site 2 of MarA (e.g., residueshistidine 54 to serine 62).

Four scaffolds were identified for MarA site 3, (e.g., residues serine55 to methionine 65):

Six scaffolds were identified for site 6 (e.g., residues leucine 24 toglutamate 35).

These scaffolds were then used to search the CambridgeSoft ACX-SCdatabase of over 600,000 non-proprietary chemical structures and thenumber of chemicals similar to the scaffolds was determined.

The term “scaffold” includes the compounds identified by the computermodeling program. These compounds may or may not be themselvestranscription factor modulating compounds. An ordinarily skilled artisanwill be able to analyze a scaffold obtained from the computer modelingprogram and modify the scaffold such that the resulting compounds haveenhanced chemical properties over the initial scaffold compound, e.g.,are more stable for administration, less toxic, have enhanced affinityfor a particular transcription factor, etc. The invention pertains notonly to the scaffolds identified, but also the transcription factormodulating compounds which are developed using the scaffolds.

Table 3 lists portions of Rob which were identified as possibleinteraction sites for a modulating compound. The invention pertains, atleast in part, to any compounds modeled to bind to these regions of Rob.The numbering corresponds to that given in SEQ ID NO. 4. TABLE 3 SiteNumber Residues (based on full length Rob) Site Label 1 37 to 45 R40Major Groove 2 43 to 54 I50 HTH Core 3 51 to 60 R55 Minor Groove 4 10 to20 W13

These scaffolds were identified as possible modulating compounds whichwith site 1 of Rob (residues 37-45), a MarA family polypeptide.

These scaffolds were identified as small molecules that may interactwith site 2 of Rob (residues 43-52), a MarA family polypeptide.

The design of compounds that bind to, modulate, or inhibit transcriptionfactors, generally involves consideration of two factors. First, thecompound must be capable of physically and structurally associating witha particular transcription factor. Non-covalent molecular interactionsimportant in the association of a transcription factor with a modulatingcompound include hydrogen bonding, van der Waals and hydrophobicinteractions.

Second, the modulating compound must be able to assume a conformationthat allows it to associate with the selected transcription factor.Although certain portions of the inhibiting compound will not directlyparticipate in this association with the transcription factor, thoseportions may still influence the overall conformation of the molecule.This, in turn, may have a significant impact on potency. Suchconformational requirements include the overall three-dimensionalstructure and orientation of the chemical entity or compound in relationto all or a portion of the binding site, e.g., active site or accessorybinding site of a particular transcription factor such as MarA, or thespacing between functional groups of a compound comprising severalchemical entities that directly interact with the particulartranscription factor.

In a further embodiment, the potential modulating effect of a chemicalcompound on a selected transcription factor (e.g., a HTH protein, a AraCfamily polypeptide, a MarA family polypeptide, e.g., MarA) is analyzedprior to its actual synthesis and testing by the use of computermodeling techniques. If the theoretical structure of the given compoundsuggests insufficient interaction and association between it and theselected transcription factor, synthesis and testing of the compound isavoided. However, if computer modeling indicates a strong interaction,the molecule may then be synthesized and tested for its ability to bindto the selected transcription factor and modulate the transcriptionfactor's activity.

A transcription factor modulating compound or other binding compound(e.g., an HTH protein modulating compound, an AraC family polypeptidemodulating compound, or a MarA family inhibiting compound, e.g., a MarAinhibiting compound) may be computationally evaluated and designed byscreening and selecting chemical entities or fragments for their abilityto associate with the individual small molecule binding sites or otherareas of a transcription factor.

One skilled in the art may use one of several methods to screen chemicalentities or fragments for their ability to associate with a selectedtranscription factor and more particularly with the individual smallmolecule binding sites of the particular transcription activationfactor. This process may begin by visually inspecting the structure ofthe transcription factor on a computer screen based on the atomiccoordinates of the transcription factor crystals. Selected chemicalentities may then be positioned in a variety of orientations, or docked,within an individual binding site of the transcription factor. Dockingmay be performed using software such as Quanta and Sybyl, followed byenergy minimization with standard molecular mechanics forcefields ordynamics with programs such as CHARMM (Brooks, B. R. et al., 1983, J.Comp. Chem., 4:187-217) or AMBER (Weiner, S. J. et al., 1984, J. Am.Chem. Soc., 106:765-784).

Specialized computer programs may also assist in the process ofselecting molecules that bind to a selected transcription factor, (e.g.,an HTH protein, an AraC family polypeptide, or a MarA familypolypeptide, e.g., MarA). The programs include, but are not limited to:

-   -   1. GRID (Goodford, P. J., 1985, “A Computational Procedure for        Determining Energetically Favorable Binding Sites on        Biologically Important Macromolecules” J. Med. Chem., 28:849-857        GRID is available from Oxford University, Oxford, UK.    -   2. AUTODOCK (Goodsell, D. S. and A. J. Olsen, 1990, “Automated        Docking of Substrates to Proteins by Simulated Annealing”        Proteins: Structure. Function, and Genetics, 8:195-202. AUTODOCK        is available from Scripps Research Institute, La Jolla, Calif.        AUTODOCK helps in docking inhibiting compounds to a selected        transcription factor in a flexible manner using a Monte Carlo        simulated annealing approach. The procedure enables a search        without bias introduced by the researcher.    -   3. MCSS (Miranker, A. and M. Karplus, 1991, “Functionality Maps        of Binding Sites: A Multiple Copy Simultaneous Search Method.”        Proteins: Structure, Function and Genetics, 11:29-34). MCSS is        available from Molecular Simulations, Burlington, Mass.    -   4. MACCS-3D (Martin, Y. C., 1992, J. Med. Chem., 35:2145-2154)        is a 3D database system available from MDL Information Systems,        San Leandro, Calif.    -   5. DOCK (Kuntz, I. D. et al., 1982, “A Geometric Approach to        Macromolecule-Ligand Interactions” J. Mol. Biol., 161:269-288).        DOCK is available from University of California, San Francisco,        Calif.        -   DOCK is based on a description of the negative image of a            space-filling representation of the molecule (i.e. the            selected transcription factor) that should be filled by the            inhibiting compound. DOCK includes a force-field for energy            evaluation, limited conformational flexibility and            consideration of hydrophobicity in the energy evaluation.    -   6. MCDLNG (Monte Carlo De Novo Ligand Generator) (D. K.        Gehlhaar, et al. 1995. J. Med. Chem. 38:466-472). MCDLNG starts        with a structure (i.e. an X-ray crystal structure) and fills the        binding site with a close packed array of generic atoms. A Monte        Carlo procedure is then used to randomly: rotate, move, change        bond type, change atom type, make atoms appear, make bonds        appear, make atoms disappear, make bonds disappear, etc. The        energy function used by MCDLNG favors the formation of rings and        certain bonding arrangements. Desolvation penalties are given        for heteroatoms, but heteroatoms can benefit from hydrogen        bonding with the binding site.

In an embodiment of the invention, docking is performed by using theAffinity program within InsightII (Molecular Simulations Inc., 1996, SanDiego, Calif., now Accelrys Inc.). Affinity is a suite of programs forautomatically docking a ligand (i.e. a transcription factor modulatingcompound) to a receptor (i.e. a transcription factor). Commands inAffinity automatically find the best binding structures of the ligand tothe receptor based on the energy of the ligand/receptor complex. Asdescribed below, Affinity allows for the simulation of flexible-flexibledocking.

Affinity consists of two commands, GridDocking and fixedDocking, underthe new pulldown Affinity in the Docking module of the Insight IIprogram. Both commands use the same, Monte Carlo type procedure to docka guest molecule (i.e. HTH protein modulating compound) to a host (i.e.,a transcription factor). They also share the feature that the “bulk” ofthe receptor (i.e. transcription factor), defined as atoms not in thebinding (active) site specified, is held rigid during the dockingprocess, while the binding site atoms and ligand atoms are movable. Thecommands differ, however, in their treatment of nonbond interactions. InGridDocking, interactions between bulk and movable atoms areapproximated by the very accurate and efficient molecularmechanical/grid (MM/Grid) method developed by Luty et al. 1995. J. Comp.Chem. 16:454, while interactions among movable atoms are treatedexactly. GridDocking also includes the solvation method of Stouten etal. 1993. Molecular Simulation 10:97. On the other hand, thefixedDocking command computes nonbond interactions using methods in theDiscover program (cutoff methods and the cell multipole method) and itdoes not include any solvation terms.

Affinity does not, generally, require any intervention from the userduring the docking. It automatically moves the ligand (i.e. modulatingcompound), evaluates energies, and checks if the structure isacceptable. Moreover, the ligand and the binding site of the receptor(i.e. the selected transcription modulator) are flexible during thesearch.

Most of the docking methods in the literature are based on descriptorsor empirical rules (for a review see Kuntz et al. 1994. Acc. Chem. Res.27:117. These include DOCK (Kuntz et al. 1982. J. Mol. Biol. 161:269.,Shoichet et al. 1992. J. Compt. Chem. 13:380., Oshiro et al. 1995. J.Comp. Aided Molec. Design 9:113.), CAVEAT (Bartlett et al. 1989.“Chemical and Biological Problems in Molecular Recognition” RoyalSociety of Chemistry: Cambridge, pp. 182-196., Lauri & Bartlett. 1994.J. Comput. Aided Mol. Design 8:51), FLOG (Miller et al. 1994. J. Comp.Aided Molec. Design 8:153), and PRO_LIGAND (Clark et al. 1995. J. Comp.Aided Molec. Design 9:13), to name a few. Affinity differs from thesemethods in several aspects.

First, it uses full molecular mechanics in searching for and evaluatingdocked structures. In contrast descriptor-based methods use empiricalrules which usually take into account only hydrogen bonding, hydrophobicinteractions, and steric effects. This simplified description ofligand/receptor interaction is insufficient in some cases. For example,Meng et al. 1992. J. Compt. Chem. 13:505 studied three scoring methodsin evaluating docked structures generated by DOCK. They found that onlythe forcefield scores from molecular mechanics correctly identifystructures closest to experimental binding geometry, while scoringfunctions that consider only steric factors or only electrostaticfactors are less successful. Note that in the study by Meng et al. 1992.J. Compt. Chem. 13:505, docking was still performed using descriptors.Affinity, on the other hand, uses molecular mechanics in both dockingand scoring and is therefore more consistent.

Second, in Affinity, while the bulk of the receptor is fixed, thedefined binding site is free to move, thereby allowing the receptor toadjust to the binding of different ligands or different binding modes ofthe same ligand. By contrast, almost all of the descriptor-based methodsfix the entire receptor.

Third, the ligand itself is flexible in Affinity which permits differentconformations of a ligand (i.e. transcription factor modulatingcompound) to be docked to a receptor (i.e. transcription factor).Recently Oshiro et al. (1995 J. Comp. Aided Molec. Design 9; 113)extended DOCK to handle flexible ligands. FLOG is also able to treatflexible ligand by including different conformations for each structurein the database (Miller et al. 1995. J. Comp. Aided Molec. Design.8:153). Most other methods are limited to rigid ligands.

There are also a few energy based docking methods (Kuntz et al. 1994.Acc. Chem Res. 27:117). These methods use either molecular dynamics(notably simulated annealing) or Monte Carlo methods. For example,Caflisch et al. 1992. Proteins: Struct. Funct. and Genetics 13:223)developed a two step procedure for docking flexible ligands. In theirprocedure, ligand is first docked using a special energy functiondesigned to remove bad contact between the ligand and the receptorefficiently. Then Monte Carlo minimization (Li & Scheraga. 1987. Proc.Natl. Acad. Sci. U.S.A. 84:6611) is carried out to refine the dockedstructures using molecular mechanics. Hart and Read. 1992. Proteins:Struct. Funct. and Genetics 13:206 also employ two steps to dockligands. They use a score function based on receptor geometry toapproximately dock ligands in the first step, and then use Monte Carlominimization similar to that of Caflisch et al. 1992. Proteins: Struct.Funct. and Genetics 13:223 for the second step. The method by Mizutaniet al. (1994. J. Mol. Biol. 243:310) is another variation of this twostep method.

Affinity uses a Monte Carlo procedure in docking ligands, but there areimportant distinctions over the prior art methods. First, the MonteCarlo procedure in Affinity can be used in conjunction either withenergy minimization (to mimic the Monte Carlo minimization method of Li& Scheraga. 1987. Proc. Natl. Acad. Sci. U.S.A. 84:6611) or withmolecular dynamics (to mimic the hybrid Monte Carlo method, Clamp et al.1994. J. Comput. Chem. 15:838, or the smart Monte Carlo method,Senderowitz et al. 1995. J. Am. Chem. Soc. 117:8211). This flexibilityallows Affinity to be applied to a variety of docking problems. Second,in the initial screening of docked structures, Affinity employs energydifferences obtained from molecular mechanics, while the methodsdiscussed above use empirical rules or descriptors. Therefore, Affinityis more consistent in that it uses molecular mechanics in both initialscreening and final refinement of docked structures. Third, Affinityallows the binding site of the receptor to relax, while the methodsdiscussed above fix the entire receptor. Fourth, Affinity employs twonew nonbond techniques which are both accurate and efficient to makedocking practical. One is the Grid/MM method of Luty et al. whichrepresents the bulk of the receptor by grids (Luty et al. 1995. J. Comp.Chem. 16:454). This method is 10-20 times faster than the no-cutoffmethod with almost no loss in accuracy. It also incorporates thesolvation method of Stouten et al. (1993. Molecular Simulation 10:97).The other is the cell multipole method. This method is about 50% slowerthan the Grid/MM method, but it does not require grid setup. Thus, atypical docking calculation takes about 1-3 hours of CPU time on anIndigo R4400 workstation.

Once suitable chemical fragments have been selected, they can beassembled into a single compound or inhibiting compound. Assembly may beproceed by visual inspection of the relationship of the fragments toeach other on a three-dimensional image display on a computer screen inrelation to the structure coordinates of a particular transcriptionfactor, e.g., MarA. This may be followed by manual model building usingsoftware such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting theindividual chemical fragments include:

-   -   1. 3D Database systems such as MACCS-3D (MDL Information        Systems, San Leandro, Calif. This area is reviewed in Martin, Y.        C., 1992, “3D Database Searching in Drug Design”, J. Med. Chem.,        35, pp. 2145-2154).    -   2. CAVEAT (Bartlett, P. A. et al, 1989, “CAVEAT: A Program to        Facilitate the Structure-Derived Design of Biologically Active        Molecules”. In Molecular Recognition in Chemical and Biological        Problems”, Special Pub., Royal Chem. Soc., 78, pp. 182-196).        CAVEAT is available from the University of California, Berkeley,        Calif. CAVEAT suggests inhibiting compounds to MarA based on        desired bond vectors.    -   3. HOOK (available from Molecular Simulations, Burlington,        Mass.). HOOK proposes docking sites by using multiple copies of        functional groups in simultaneous searches.

In another embodiment, transcription factor modulating compounds may bedesigned as a whole or “de novo” using either an empty active site oroptionally including some portion(s) of a known inhibiting compound(s).These methods include:

-   -   1. LUDI (Bohm, H.-J., “The Computer Program LUDI: A New Method        for the De Novo Design of Enzyme Inhibiting compounds”, J. ComR.        Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available from        Biosym Technologies, San Diego, Calif. LUDI is a program based        on fragments rather than on descriptors. LUDI proposes somewhat        larger fragments to match with the interaction sites of a        macromolecule and scores its hits based on geometric criteria        taken from the Cambridge Structural Database (CSD), the Protein        Data Bank (PDB) and on criteria based on binding data. LUDI is a        library based method for docking fragments onto a binding site.        Fragments are aligned with 4 directional interaction sites        (lipophilic-aliphatic, lipophilic-aromatic, hydrogen donor, and        hydrogen acceptor) and scored for their degree of overlap.        Fragments are then connected (i.e. a linker of the proper length        is attached to each terminal atom in the fragments). Note that        conformational flexibility can be accounted for only by        including multiple conformations of a particular fragment in the        library.    -   2. LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985        (1991)). LEGEND is available from Molecular Simulations,        Burlington, Mass.    -   3. CoMFA (Conformational Molecular Field Analysis) (J. J.        Kaminski. 1994. Adv. Drug Delivery Reviews 14:331-337.) CoMFA        defines 3-dimensional molecular shape descriptors to represent        properties such as hydrophobic regions, sterics, and        electrostatics. Compounds from a database are then overlaid on        the 3D pharmacophore model and rated for their degree of        overlap. Small molecule databased that be searched include: ACD        (over 1,000,000 compounds), Maybridge (about 500,000 compounds),        NCI (about 500,000 compounds), and CCSD. In measuring the        goodness of the fit, molecules can either be fit to the 3D        molecular shape descriptors or to the active conformation of a        known inhibiting compound.    -   4. LeapFrog (available from Tripos Associates, St. Louis, Mo.).

FlexX (© 1993-2002 GMD German National Research Center for InformationTechnology; Rarey, M. et al J. Mol. Biol., 261:407-489) is a fast,flexible docking method that uses an incremental construction algorithmto place ligands into and active site of the transcription factor.Ligands (e.g., transcription factor modulating compounds) that arecapable of “fitting” into the active site are then scored according toany number of available scoring schemes to determine the quality of thecomplimentarily between the active site and ligand.

Other molecular modeling techniques may also be employed in accordancewith this invention. See, e.g., Cohen, N. C. et al., “Molecular ModelingSoftware and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp.883-894 (1990). See also, Navia, M. A. and M. A. Murcko, “The Use ofStructural Information in Drug Design”, Current Opinions in StructuralBiology, 2, pp. 202-210 (1992).

Candidate transcription factor modulating compounds can be evaluated fortheir modulating, e.g., inhibitory, activity using conventionaltechniques which may involve determining the location and bindingproximity of a given moiety, the occupied space of a bound inhibitingcompound, the deformation energy of binding of a given compound andelectrostatic interaction energies. Examples of conventional techniquesuseful in the above evaluations include, but are not limited to, quantummechanics, molecular dynamics, Monte Carlo sampling, systematic searchesand distance geometry methods (Marshall, G. R., 1987, Ann. Ref:Pharmacol. Toxicol., 27:193). Examples of computer programs for suchuses include, but are not limited to, Gaussian 92, revision E2(Gaussian, Inc. Pittsburgh, Pa.), AMBER version 4.0 (University ofCalifornia, San Francisco), QUANTA/CHARMM (Molecular Simulations, Inc.,Burlington, Mass.), and Insight II/Discover (Biosym Technologies Inc.,San Diego, Calif.). These programs may be implemented, for example,using a Silicon Graphics Indigo2 workstation or IBM RISC/6000workstation model 550. Other hardware systems and software packages willbe known and of evident applicability to those skilled in the art.

Once a compound has been designed and selected by the above methods, theefficiency with which that compound may bind to a particulartranscription factor may be tested and optimized by computationalevaluation. An effective transcription factor modulating compound shoulddemonstrate a relatively small difference in energy between its boundand free states (i.e., a small deformation energy of binding).Transcription factor modulating compounds may interact with the selectedtranscription factor in more than one conformation that is similar inoverall binding energy. In those cases, the deformation energy ofbinding may be taken to be the difference between the energy of the freecompound and the average energy of the conformations observed when theinhibiting compound binds to the enzyme.

A compound designed or selected as interacting with a selectedtranscription factor, e.g., a MarA family polypeptide, e.g., MarA, maybe further computationally optimized so that in its bound state it wouldpreferably lack repulsive electrostatic interaction with the targetenzyme. Such non-complementary (e.g., electrostatic) interactionsinclude repulsive charge-charge, dipole-dipole and charge-dipoleinteractions. Specifically, the sum of all electrostatic interactionsbetween the modulating compound and the enzyme when the modulatingcompound is bound to the selected transcription factor, preferably makea neutral or favorable contribution to the enthalpy of binding.

Specific computer software is available in the art to evaluate compounddeformation energy and electrostatic interaction. Examples of programsdesigned for such uses include: Gaussian 92, revision C [M. J. Frisch,Gaussian, Inc., Pittsburgh, Pa. © 1992]; AMBER, version 4.0 [P. A.Kollman, University of California at San Francisco, © 1994];QUANTA/CHARMM [Molecular Simulations, Inc., Burlington, Mass. © 1994];and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif.(1994). These programs may be implemented, for instance, using a SiliconGraphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550.Other hardware systems and software packages will be known to thoseskilled in the art.

Once a transcription factor modulating compound has been optimallyselected or designed, as described above, substitutions may then be madein some of its atoms or side groups in order to improve or modify itsbinding properties. Initial substitutions are preferable conservative,i.e., the replacement group will have approximately the same size,shape, hydrophobicity and charge as the original group. Substitutionsknown in the art to alter conformation should be avoided. Suchsubstituted chemical compounds may then be analyzed for efficiency offit to the selected transcription factor by the same computer methodsdescribed above.

Computer programs can be used to identify unoccupied (aqueous) spacebetween the van der Waals surface of a compound and the surface definedby residues in the binding site. These gaps in atom-atom contactrepresent volume that could be occupied by new functional groups on amodified version of the lead compound. More efficient use of theunoccupied space in the binding site could lead to a stronger bindingcompound if the overall energy of such a change is favorable. A regionof the binding pocket which has unoccupied volume large enough toaccommodate the volume of a group equal to or larger than a covalentlybonded carbon atom can be identified as a promising position forfunctional group substitution. Functional group substitution at thisregion can constitute substituting something other than a carbon atom,such as oxygen. If the volume is large enough to accommodate a grouplarger than a carbon atom, a different functional group which would havea high likelihood of interacting with protein residues in this regionmay be chosen. Features which contribute to interaction with proteinresidues and identification of promising substitutions includehydrophobicity, size, rigidity and polarity. The combination of docking,K_(i) estimation, and visual representation of sterically allowed roomfor improvement permits prediction of potent derivatives.

Similarity Screening

Once a transcription factor modulating compound has been selected ordesigned, computational methods to assess its overall likeness orsimilarity to other molecules can be used to search for additionalcompounds with similar biochemical behavior. In such a way, forinstance, HTS derived hits can be tested to assure that they are bonafide ligands against a particular active site, and to eliminate thepossibility that a particular hit is an artifact of the screeningprocess. There are currently several methods and approaches to determinea particular compound's similarity to members of a virtual database ofcompounds. One example is the OPTISIM methodology that is distributed inthe Tripos package, SYBYL (© 1991-2002 Tripos, Inc., St. Louis, Mo.).OPTISIM exploits the fact that each 3-dimensional representation of amolecule can be broken down into a set of 2-dimensional fragments andencoded into a pre-defined binary string. The result is that eachcompound within a particular set is represented by a unique numericalcode or fingerprint that is amenable to mathematical manipulations suchas sorting and comparison. OPTISIM is automated to calculate and reportthe percent difference in the fingerprints of the respective compoundsfor instance according to the using a formalism known as the Tanimotocoefficient. For instance, a compound that is similar in structure toanother will share a high coefficient. Large virtual databases ofcommercially available compounds or of hypothetical compounds can bequickly screened to identify compounds with high Tanimoto coefficient.

CoMFA/QSAR

Once a series of similar transcription factor modulating compounds hasbeen identified and expanded by the methods described, theirexperimentally determined biological activities can be correlated withtheir structural features using a number of available statisticalpackages. In a typical project within the industry, the CoMFA(COmparative Molecular Field Analysis) and QSAR (Quantitative StructureActivity Relationship) packages within the SYBYL suite of programs(Tripos Associates, St. Louis, Mo.) are utilized. In CoMFA, a particularseries of compounds with measured activities are co-aligned in a mannerthat is believed to emulate their arrangement as they interact with theactive site. A 3-dimensional lattice, or grid is then constructed toencompass the collection of the so-aligned compounds. At each point onthe lattice, an evaluation of the potential energy is determined andtabulated-typically potentials that simulate the electronic and stericfields are determined, but other potential functions are available.Using the statistical methods such as PLS (Partial Least Squares),correlation between the measured activities and the potential energyvalues at the grid-points can be determined and summed in a linearequation to produce the overall molecular correlation or QSAR model. Aparticularly useful feature in CoMFA is that the individual contributionfor each grid-point is known; the importance of the grid points upon theoverall correlation can be visualized graphically in what is referred toas a CoMFA field. When this field is combined with the original compoundalignment, it becomes a powerful tool to rationalize the activities ofthe individual compounds from whence the model was derived, and topredict how chemical modification of a reference compound would beeffected. As an example, a QSAR model was developed for a set of 92benzodiazepines using the method described above. A representative CoMFAfield is shown in FIG. 4; the region delineated by wire mesh (adjacentto the referenced triazinoxazepine) is the region where chemicalmodification characterized by increasing steric bulk would lead tofavorable effects in transcription factor modulation.

The invention pertains, per se, to not only the methods for identifyingthe transcription factor modulating compounds, but to the compoundsidentified by the methods of the invention as well as methods for usingthe identified compounds.

VIII. MarA Family Modulating Compounds, and Methods of Use Thereof.

In an embodiment, the invention pertains to methods for modulating atranscription factor, e.g., an HTH protein, an AraC family polypeptide,or a MarA family polypeptide. The method includes contacting thetranscription factor, e.g., a MarA family polypeptide, with atranscription factor modulutating compound of the formula (I):A-E  (I)

wherein A is a polar moiety, E is a hydrophobic moiety, andpharmaceutically acceptable salts thereof. The transcription factormodulating compound, e.g., a MarA family modulating compound, maycomprise one or more polar moieties and/or one or more hydrophobicmoieties.

In another embodiment, the invention pertains to methods for reducingantibiotic resistance of a microbial cell. The method includescontacting the cell with a transcription factor modulating compound,e.g., a MarA family modulating compound, such that the antibioticresistance of the cell is reduced.

In another embodiment, the invention pertains to inhibitingtranscription, comprising contacting a transcription factor with atranscription factor modulating compound, such that transcription isinhibited. In a further embodiment, the transcription of a prokaryoticcell is inhibited. In another further embodiment, the transcriptionfactor modulating compound is a compound of anyone of formulae(I)-(XVII).

The term “antibiotic resistance” includes resistance of a microbial cellto a antibiotic compound, especially an antibiotic compound which hadbeen previously used to treat similar microbial organisms successfully.

The term “polar moiety” includes moieties with at least one heterocycle.It also includes moieties such as, but not limited to, hydroxyl,halogens, thioethers, carboxylic acids, metals (e.g. alkali, alkaline,Au, Hg, Ag, Mn, Co, Cu, Zn, etc.), nitro, amino, alkoxy, and othermoieties which allow the compound to perform its intended function. Theterm “polar moiety” includes moieties which allow the transcriptionfactor modulating compound to perform its intended function, e.g.,modulate a transcription factor, e.g., an AraC family polypeptide or aMarA family polypeptide. A heterocyclic polar moiety may comprise one ormore rings, one or more of which may be aromatic. In an embodiment, oneor more rings of the polar moiety are fused. The heterocyclic polarmoiety may also be bicyclic.

The heterocyclic polar moiety may comprise one or more nitrogen, sulfur,or oxygen atoms. Examples of heterocycles include benzodioxazole,benzofuran, benzoimidazole, benzoxazole, benzothiazole, benzothiophene,chromenone, deazapurine, furan, imidazole, imidazopyridine, indole,indolizine, isooxazole, isothiaozole, isoquinoline,methylenedioxyphenyl, napthridine, oxazole, purine, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrollidine, quinoline,tetrazole, thiazole, thiophene, triazole, and triazoletetrazole.

Furthermore, the polar moiety may be substituted when chemicallyfeasible. For example, the polar moiety may be substituted with one ormore substituents such as alkyl, alkenyl, alkynyl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.Examples of substituents also include nitro, alkoxy, aryl, amidyl,ester, thioester, alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl,etc.), araalkyl (e.g., substituted or unsubstituted benzyl), hydroxy,halogen (e.g., fluorine, bromine, chlorine, iodine, etc.).

The term “hydrophobic moiety” includes moieties such as which allow thetranscription factor modulating compound (e.g., an HTH proteinmodulating compound, an AraC family polypeptide modulating compound, aMarA family polypeptide modulating compound, etc.) to perform itsintended function, e.g., modulate a transcription factor. Examples ofhydrophobic moieties include, for example, hydrogen, alkyl, alkenyl,alkynyl, and aryl moieties. The hydrophobic moieties may beunsubstituted or substituted, if chemically feasible (e.g., nothydrogen). In an embodiment, the hydrophobic moiety is substituted orunsubstituted phenyl. Examples of substituents include alkyl, alkenyl,alkynyl, alkoxy, halogen, amino, thiol, hydroxy, nitro, aryl, andheteroaryl. The substituents can be substituted or unsubstituted. In anembodiment, the phenyl hydrophobic moiety is para-substituted, e.g.,alkyl (methyl, ethyl, propyl, butyl, pentyl, etc.), halogen (e.g.,fluorine, bromine, chlorine, iodine, etc.), hydroxy, substituted.

In another embodiment, the hydrophobic moiety is heterocyclic. Examplesof heterocyclic hydrophobic moieties include imidazopyridine,quinolinyl, pyridinyl, etc.

In one embodiment, the transcription factor modulating compound (e.g.,MarA family polypeptide modulating compound, AraC family polypeptidemodulating compound, etc.) is of the formula (VII):

wherein

W is NH, O or S;

X is O, S, or C, optionally linked to Q;

A¹ is C-Z¹, O, or S;

A² is C-Z², O, or S;

A³ is C-Z³, O, or S;

A⁴ is C-Z⁴, O, or S;

A⁵ is C-Z⁵, or N-Z⁵;

Z¹, Z², Z³, and Z⁴ are each independently selected from the groupconsisting of hydrogen, alkoxy, hydroxy, halogen, and alkyl;

Z⁵ is hydrogen, alkoxy, hydroxy, halogen, alkyl, or carbonyl;

Q is hydrogen, alkyl, alkenyl, alkynyl, halogen, hydroxy, aryl, andpharmaceutically acceptable salts thereof.

In yet another embodiment, the transcription factor modulating compound(e.g., the MarA family polypeptide modulating compound, AraC familypolypeptide modulating compound, etc.) is of the formula (II):

wherein

W is O or S;

X is O, S, or C, optionally linked to Q;

A¹ is C-Z⁴, O, or S;

A² is C-Z⁵, or N-Z⁵;

Z¹, Z², Z³, Z⁴ and Z⁵ are each independently hydrogen, alkoxy, hydroxy,halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, or cyano;

Z³ is hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl,heterocyclic, amino, nitro, cyano, carbonyl, or thiocarbonyl;

Q is an aromatic or heterocyclic moiety, and pharmaceutically acceptablesalts thereof.

In a further embodiment, W may be oxygen and X may be oxygen.Furthermore, A¹ and A² may be C-Z⁴ and C-Z⁵, respectively. Examples ofZ⁴ and Z⁵ include hydrogen and hydroxy. Examples of Z¹ and Z² includehydrogen and hydroxy. Other examples of Z² also include halogen, e.g.,fluorine, chlorine, bromine, and iodine. Examples of Z³ include, forexample, hydrogen, alkoxy and hydroxy. Examples of Q include substitutedand unsubstituted phenyl. The phenyl may be para-substituted. Examplesof substituents include hydroxyl, halogen (e.g., fluorine, bromine,chlorine, iodine, etc.), amino, alkyl (e.g., methyl, ethyl, propyl,butyl, pentyl, etc.), nitro, cyano, etc. In an embodiment, thetranscription factor modulating compound is a MarA modulating compound,and in a further embodiment, a MarA inhibiting compound.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (VIII):

wherein:

G is a substituted or unsubstituted aromatic moiety, heterocyclic,alkyl, alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, orhydrogen;

L¹, L², L³, L⁴, L⁹ and L¹⁰ are each independently oxygen, sulfur,substituted or unsubstituted nitrogen, and substituted or unsubstitutedcarbon; and

L⁵ and L⁶ are each independently hydrogen, substituted or unsubstitutedalkyl, alkenyl, alkynyl, acyl, heterocyclic, amino, nitro, hydroxy,cyano, alkoxy, or aryl, and L⁵ and L⁶ may optionally be linked with achain of one to six atoms to form a fused ring, and pharmaceuticallyacceptable salts thereof.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (IX):

wherein:

G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl,alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;

L¹, L², L³, and L⁴ are each independently oxygen, sulfur, substituted orunsubstituted nitrogen, and substituted or unsubstituted carbon; and

R⁹, L⁵ and L⁶ are each independently hydrogen, substituted orunsubstituted alkyl, alkenyl, alkynyl, acyl, heterocyclic, amino, nitro,hydroxy, cyano, alkoxy, or aryl, and L₅ and L₆ may optionally be linkedwith a chain of one to six atoms to form a fused ring, andpharmaceutically acceptable salts thereof.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (III):

wherein

G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl,alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;and

L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, and L¹⁰ are each independentlyoxygen, substituted or unsubstituted nitrogen, sulfur and or substitutedor unsubstituted carbon, and pharmaceutically acceptable salts thereof.

In a further embodiment, L⁹ is N—R⁹, wherein R⁹ is hydrogen, substitutedor unsubstituted alkyl, alkenyl, alkynyl, acyl, or aryl. In another, L¹⁰is oxygen. In an embodiment, R⁹ is hydrogen. In another, G issubstituted or unsubstituted phenyl or heteroaryl. In a furtherembodiment, G is cycloalkenyl, e.g., cyclohexenyl. In one embodiment,L¹, L², L³, and L⁴ are each substituted or unsubstituted carbon and L⁵,L⁶, and L⁸ are each nitrogen. L⁷ may be substituted carbon, e.g.,substituted with a thioether moiety. In another embodiment, L⁹ and L¹⁰are each nitrogen. In another embodiment, the invention pertains tocompounds of formula (III), wherein L⁹ is nitrogen, L¹⁰ is oxygen, L¹-L⁸are each C—H, the dotted line represents a double bond and where G isnot hydrogen or methyl.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (X):

wherein

Y¹ and Y² are each oxygen, sulfur, or substituted or unsubstitutedcarbon;

J¹, J², J³, and J⁴ are each oxygen, nitrogen, or optionally substitutedcarbon, and pharmaceutically acceptable salts thereof.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (IV):

wherein

Y¹ and Y² are each oxygen or sulfur;

J is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl,cyano, nitro, amino, or halogen;

V is substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy,alkylamino, or alkylthio;

P and K are each independently substituted or unsubstituted aryl, andpharmaceutically acceptable salts thereof.

In a further embodiment, Y¹ and Y³ are each oxygen, V is alkoxy and J islower alkyl. In another embodiment, P is substituted or unsubstitutedphenyl. K may be substituted or unsubstituted heteroaryl.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (V):

wherein

T¹, T², T³, T⁴, T⁵, and T⁶ are each independently substituted orunsubstituted carbon, oxygen, substituted or unsubstituted nitrogen, orsulfur;

M is hydrogen, alkyl, alkenyl, alkynyl, heterocyclic or aryl, orpharmaceutically acceptable salts thereof.

In a further embodiment, T⁵ is N—W or C—W, wherein W is alkyl, alkenyl,alkynyl, aryl, heterocyclic, acyl, hydroxy, alkoxy, alkthio, amino,nitro, halogen, or hydrogen. In another further embodiment, T⁶ is N.

In a further embodiment, M is substituted or unsubstituted aryl. W maybe substituted or unsubstituted alkyl. In another embodiment, T¹, T², T³and T⁴ are each substituted or unsubstituted carbon. In a furtherembodiment, at least one of T¹, T², T³, and T⁴ is nitrogen, and theremaining T moieties are substituted or unsubstituted carbon.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (Va):

wherein

R¹ is OH, OCOCO₂H, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group;

R² is H, CO₂(C₁-C₅ substituted or unsubstituted, straight or branchedalkyl), or a substituted or unsubstituted aryl group; and

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen.

In certain embodiments of formula Va, those compounds disclosed in U.S.Ser. No. 10/139,591, filed May 6, 2002, are excluded from the scope ofthe present invention.

In other embodiments of formula Va, when R⁶ is NO₂ and R² isunsubstituted phenyl, then R¹ is not O(CHCH₃)(CO₂)CH₂CH₃ or OCH₂CO₂H.Also, in another embodiment, when R⁶ is H or NO₂, then R¹ is not aphenyl-substituted alkyloxy group. In yet another embodiment, when R⁴,R⁵, R⁶, and R⁷ are all H and R² is para-methoxyphenyl, then R¹ is notOH. And in another embodiment, when R⁴, R⁵, R⁶, and R⁷ are all H and R²is unsubstituted phenyl, then R¹ is not OCH₂CO₂CH₂CH₃;

In certain aspects of formula Va, R⁴, R⁵, and R⁷ are all H.

Similarly, R¹ of formula Va may be selected from the group consisting ofOH, O(CR′R″)₁₋₃H, O(CR′R″)₁₋₃OH, O(CR′R″)₁₋₃CO₂H,O(CR′R″)₁₋₃CO₂(CR′R″)₁₋₃H, O(CR′R″)₁₋₃(CO)NH₂, O(CR′R″)₁₋₃(CNH)NH₂,OCOCO₂H, O(CR′R″)₁₋₃SO₃H, O(CR′R″)₁₋₃OSO₃H, O(CR′R″)₁₋₃PO₃H,O(CR′R″)₁₋₃OPO₃H, O(CR′R″)₁₋₃N[(CR′R″)₀₋₃H]₂, O(CR′R″)₁₋₃(CO)(NHOH), andO(CR′R″)₁₋₃(heteroaryl); wherein R′ and R″ are each independently H, aC₁-C₃ alkyl, C₂-C₃ alkenyl, or C₂-C₃ alkynyl group. Each R′ and R″ ispreferably H or CH₃.

When R¹ of formula Va is O(CR′R″)₁₋₃(heteroaryl), the heteroaryl groupmay be a pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl,imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl,pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.

Similarly, when R² of formula Va may be a substituted or unsubstitutedphenyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl,imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl,pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.

In a more particular embodiment, R⁶ of formula Va is H, (CR′R″)₁₋₃H,(CR′R″)₁₋₃OH, (CR′RC″)₁₋₃NH₂, (NOH)(CR′R″)₁₋₃H, CO(CR′R″)₁₋₃NH₂,CO(CR′R″)₁₋₃H, CO(CR′R″)₁₋₃OH, CO(CR′R″)₀₋₃CF₃,(CR′R″)₀₋₃N[(CR′R″)₀₋₃H]₂, CO(substituted or unsubstituted heteroaryl),CO(C₃-C₆ substituted or unsubstituted cycloalkyl), O(CR′R″)₁₋₃H,CO(substituted or unsubstituted phenyl), CO₂(CR′R″)₁₋₃H, CN, NO₂, F, Cl,Br, or I, wherein R′ and R″ are each independently H, a C₁-C₃ alkyl,C₂-C₃ alkenyl, or C₂-C₃ alkynyl group. Preferably each R′ and R″ isindependently H or CH₃.

In yet another embodiment, R⁶ of formula Va is CO(substituted orunsubstituted heteroaryl), wherein said heteroaryl group is a pyrrolyl,furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl,tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl,pyridazinyl, or pyrimidinyl group.

In another embodiment, R⁴, R⁵, and R⁷ are each hydrogen; R⁶ is NO₂, andR¹ is hydroxyl. In a further embodiment, R² is substituted aryl, e.g.,substituted phenyl, substituted furanyl, or substituted benzoimidazole.In a further embodiment, when R² is substituted phenyl, R² issubstituted with an optionally substituted arylcarbonylamino group, anamino group, a dialkyl amino group, or a carboxylate group. The arylcarbonylamino group may be substituted with dialkyl amino, alkyl, orhalogens. In a further embodiment, when R² is a substituted furanylgroup, R² is substituted with an aryl group, e.g., phenyl. In anotherembodiment, when R² is an optionally substituted benzoimidazole, it issubstituted with an alkyl group.

In another embodiment, the transcription factor modulating compound isof the formula (XI):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R^(21a) and R^(21b) are independently selected from the group consistingof H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁵, R⁷ andR²⁶ can be H. In yet another embodiment, R⁶ may be NO₂. In yet anotherembodiment, R²⁵ can be a substituted alkenyl group, wherein saidsubstituted alkenyl group is substituted with substituted orunsubstituted phenyl. Suitable substituted phenyl groups include, forexample, para-halogenated phenyl groups, such as para-fluoro phenyl.

In another embodiment, the invention provides transcription factormodulating compounds of the formula (XII):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R²² is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, aroyl and pharmaceuticallyacceptable salts, esters and prodrugs thereof.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁵, R⁷ andR²⁶ can be H. In yet another embodiment, R⁶ may be NO₂. In yet anotherembodiment, R²⁵ can be a substituted alkenyl group, wherein saidsubstituted alkenyl group is substituted with substituted orunsubstituted phenyl. Suitable substituted phenyl groups include, forexample, para-halogenated phenyl groups, such as para-fluoro phenyl.

In another embodiment, the invention provides transcription factormodulating compounds of the formula (XIII):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R²³ and R²⁴ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino; aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof;

provided that when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, thenR²³ is not methyl, unsubstituted phenyl, or unsubstituted furanyl.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁵, R⁷ andR²⁶ can be H. In yet another embodiment, R⁶ may be NO₂. In yet anotherembodiment, R²⁵ can be a substituted alkenyl group, wherein saidsubstituted alkenyl group is substituted with substituted orunsubstituted phenyl. Suitable substituted phenyl groups include, forexample, para-halogenated phenyl groups, such as para-fluoro phenyl.

In another embodiment, the invention provides a method for reducingantibiotic resistance of a microbial cell, comprising contacting saidcell with a transcription factor modulating compound of the formula(XIV):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R²⁵ and R²⁶ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof;

provided that when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, thenR²⁵ is not unsubstituted phenyl or O-tert-butyl.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁵, R⁷ andR²⁶ can be H. In yet another embodiment, R⁶ may be NO₂. In yet anotherembodiment, R²⁵ can be a substituted alkenyl group, wherein saidsubstituted alkenyl group is substituted with substituted orunsubstituted phenyl. Suitable substituted phenyl groups include, forexample, para-halogenated phenyl groups, such as para-fluoro phenyl.

In another embodiment, the invention provides a transcription factormodulating compound of the formula (XV):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶ and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen;

R²⁷ is selected from the group consisting of substituted heteroaryl;substituted alkyl; substituted or unsubstituted alkenyl; alkynyl;alkylcarbonyl, arylcarbonyl; heteroarylcarbonyl; sulfonyl; alkylamino;arylamino; heteroarylamino; alkoxy, aryloxy, heteroaryloxy; substitutedstraight chain C₁-C₅ alkyl or alkenyl; substituted or unsubstitutedisoxazole, thiazolidine, imidazole, quinoline, pyrrole, triazole, orpyrazine; 2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl,and 1-fluorophenyl; and

R²⁸ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, aroyl and pharmaceuticallyacceptable salts, esters and prodrugs thereof.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁵, R⁷ andR²⁸ may each be H. In a further embodiment, R⁶ can be NO₂.

In one embodiment, R²⁷ may be substituted alkyl which can be substitutedwith, for example, substituted or unsubstituted phenyl. In anotherembodiment, said substituted phenyl may be substituted with alkoxy, suchas para-alkoxy phenyl. In a particular embodiment, the para-alkoxyphenyl can be para-methoxy phenyl.

In another embodiment, R²⁷ may be a meta-substituted phenyl, whereinsaid meta-substituted phenyl can be alkyl substituted. In a particularembodiment, the meta-substituted phenyl can be meta-methyl phenyl. Inanother particular embodiment, meta-substituted phenyl may be meta-cyanophenyl. In a further embodiment, the meta-substituted phenyl can besubstituted with a halogen. Furthermore, the meta-substituted phenyl maybe, for example, meta-fluoro phenyl.

In yet another embodiment, R²⁷ can be a heteroaryl group. Suitableheteroaryl groups include, for example, methyl-pyrrolyl and furanyl.

In amother embodiment, the invention provides a transcription factormodulating compound of the formula (XVI):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶ and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen;

R²⁹, R³⁰ and R³¹ are independently selected from the group consisting ofH, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl, andpharmaceutically acceptable salts, esters and prodrugs thereof.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁵, R⁷ andR³¹ may each be H. In a further embodiment, R⁶ may be NO₂.

In another embodiment, R³⁰ can be H. In a further embodiment, R²⁹ may besubstituted or unsubstituted phenyl, wherein said substituted phenyl canbe substituted with alkoxy. In a particular embodiment, said substitutedphenyl can be, for example, ortho-alkoxy substituted phenyl.Furthermore, said substituted phenyl can be ortho-methoxy phenyl.

In yet another embodiment, R²⁹ may be H. In a further embodiment, R³⁰may be substituted alkenyl, wherein said substituted alkenyl can besubstituted with a substituted or unsubstituted phenyl. Furthermore, thesubstituted phenyl can be para-alkyl phenyl, para-alkoxy phenyl,ortho-alkoxy phenyl or a halogenated phenyl. In addition, thesubstituted phenyl group can be, for example, para-methyl phenyl,para-methoxy phenyl, ortho-methoxy phenyl, para-cyano phenyl,para-trifluoromethyl phenyl, para-fluoro phenyl, ortho, para-difluorophenyl or meta, para-difluoro phenyl.

In yet a further embodiment, R³⁰ can be a heteroaryl group, wherein saidheteroaryl group can be furanyl.

The invention also includes a transcription factor modulating compoundof the formula (XVII):

wherein

R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight or branchedC₁-C₅ alkyloxy group, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyl group;

R⁴, R⁵, R⁶ and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen;

R³² is selected from the group consisting of OH, Br, CN, CO₂H,morpholinyl, substituted aryl, substituted or unsubstituted alkenyl,alkynyl, heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl,arylsulfonyl, aminosulfonyl, alkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, acyl, acylamino, alkylamino, dialkylamino,arylamino, heteroarylamino, aroyl;

R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof;

provided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is NO₂, thenR³² is not dimethylamino;

provided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is Br, thenR³² is not dimethylamino.

In one embodiment, R¹ may be OH. In another embodiment, R⁴, R⁷ and R³³may each be H. In a further embodiment, R⁵ can be H. In yet anotherembodiment, R⁶ can be NO₂. In another embodiment, R³² can be a carbonylgroup, such as an aldehyde or an acylcarbonyl. In a further embodiment,R³² may be CN. In a further embodiment, R³² can be a heteroaryl group,wherein said heteroaryl group may be oxazolyl or triazolyl. In anotherembodiment, R⁶ may be CN or dialkylamino and R³² may be dialkylamino. Ina further embodiment, R⁶ can be H, R⁵ can be CN and R³² can bedialkylamino.

In a further embodiment, the transcription factor modulating compound isof the formula (Va):

wherein

R¹ is OH, or a substituted or unsubstituted straight or branchedalkyloxy group;

R² is a substituted or unsubstituted aryl or heteroaryl group;

R⁴, R⁵, and R⁷ are independently selected from the group consisting ofH, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and

R⁶ is an electron withdrawing substitutent;

provided that when R⁶ is NO₂ and R² is unsubstituted phenyl, then R¹ isnot O(CHCH₃)(CO₂)CH₂CH₃ or OCH₂CO₂H;

provided that when R⁶ is H or NO₂, then R¹ is not a phenyl-substitutedalkyloxy grou, and pharmaceutically acceptable salts, esters, andprodrugs thereof.

In another embodiment, R⁶ is an electron withdrawing substituent.Examples of electron withdrawing substituted include halogens (e.g., F,Cl, Br, etc.), halogenated alkyls (e.g., CF₃, CF₂CF₃, etc.), NO₂,C(NOH)(CR′R″), wherein each R′ and R″ are each independently H or loweralkyl (e.g., CH₃, ethyl, propyl, butyl, etc.).

In a further embodiment, R⁴, R⁵, and R⁷ are each H.

Examples of R¹ include OH, substituted or unsubstituted alkoxy (e.g.,OCH₃, OCH₂CN).

In another embodiment, R² is substituted or unsubstituted aryl. Examplesinclude phenyl, furanyl, or benzimidazolyl. In a further embodiment,wherein R² substituted phenyl which is substituted by arylcarbonylamino,heteroarylcarbonylamino, alkylcarbonyl, alkyloxy, amino or dialkylamino.In a further embodiment, R² is para-arylcarbonylamino phenyl,ortho-heteroarylcarbonylamino phenyl, para-alkylcarbonylamino phenyl,para- and ortho-alkyloxy phenyl, para-amino phenyl, meta-amino phenyl,para-dialkylamino phenyl, or meta-dialkylamino phenyl.

In a further embodiment, R² is substituted furanyl (e.g., substitutedaryl 2-furanyl, alkyl 2-furanyl). In a further embodiment, R² is asubstituted benzimidazolyl, e.g., 1-benzimidazolyl substituted byCH₂CO₂H.

In a further embodiment, the transcription factor modulating compoundis:

The EC₅₀ of a transcription factor modulating compound can be measuredusing the assay described in Example 12. In a further embodiment, thetranscription factor modulating compound has an EC₅₀ activity againstSoxS of less than about 100M, less than about 5 μM or less than about 1μM. In a further embodiment, the transcription factor modulatingcompound can have an EC₅₀ activity against MarA of less than about 10μM, less than about 5 μM, or less than about 1 μM. In yet anotherembodiment, the transcription factor modulating compound can have anEC₅₀ against LcrF (VirF) of less than about 10 μM, less than about 5 μM,or less than about 1 μM.

In another further embodiment, the transcription factor modulatingcauses a log decrease in CFU/g of kidney tissue. This can be measuredusing the assay described Example 13. In one embodiment, thetranscription factor modulating compound cause a log decrease in CFU/gof kidney tissue of greater than 1.0 CFU/g. In a further embodiment, thecompound causes a log decrease in CFU/g of kidney tissue greater than2.5 CFU/g.

In another embodiment, the transcription factor modulating compound(e.g., an AraC family polypeptide modulating compound, a MarA familypolypeptide modulating compound, etc.) is of the formula (VI):

wherein

G¹, G², and G³ are each independently O, S, substituted or unsubstitutednitrogen, or substituted or unsubstituted carbon;

E¹, E², and E³ are each independently hydrogen, alkyl, alkenyl, alkynyl,aryl, aralkyl, or acyl; and

E⁴ is alkyl, alkenyl, alkynyl, aryl, halogen, cyano, amino, nitro, oracyl, and pharmaceutically acceptable salts thereof.

In a further embodiment, G¹, G² and G³ are each oxygen.

Other transcription factor modulating compounds are shown in Table 3.The invention pertains to each of these compounds, methods (boththerapeutic and otherwise) using each of the compounds, and compositionscomprising at least one of the compounds of Table 4, Table 5, Table 6,Table 7, Table 8, Table 9, Table 10, or Table 11 or of formulae (I),(II), (III), (IV), (V), (Va), (VI), (VII), (VIII), (IX), (X), (XI),(XII), (XIII), (XIV), (XV), (XVI), (XVII).

The invention also pertains to each of the following compounds:2-(4-isopropylphenyl)-4H-chromen-4-one;2-(3,4-Dihydroxy-phenyl)-3,5,7-trihydroxy-chromen-4-one;N-isopropyl-2-[(4-methyl-5-quinolin-6-yl-4H-1,2,4-triazol-3-yl)thio]acetamide;4-hydroxy-6-methyl-5,6-dihydro-2H-pyrano[3,2-c]quinoline-2,5-dione;5,7-Dihydroxy-2-(4-hydroxy-phenyl)-chromen-4-one;2-[4-(dimethylamino)phenyl]-4H-chromen-4-one;1-(benzyloxy)-2-phenyl-1H-imidazo[4,5-b]pyridine;2-(benzylthio)-4-phenyl-5-(1-phenyl-1H-1,2,3,4-tetraazol-5-yl)pyrimidine;6-fluoro-2-phenyl-4H-chromen-4-one; 7-methoxy-2-phenyl-4H-chromen-4-one;4-(1,3-dioxo-1,3-dihydro-2H-inden-2-yliden)-2-phenyl-6-(2-pyridinyl)tetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione;2-(2-Hydroxy-3-oxo-5-p-tolyl-2,3-dihydro-furan-2-yl)-malonamic acidethyl ester; 2-[(6-nitro-2-phenyl-1H-1,3-benzimidazol-1-yl)oxy]aceticacid; 2-(4-fluorophenyl)-4H-chromen-4-one;1-methoxy-2-(4-methylphenyl)-1H-imidazo[4,5-b]pyridine;6-(5-Iodo-furan-2-yl)-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;6-(4-Ethoxy-phenyl)-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;3-Methylsulfanyl-6-(5-nitro-furan-2-yl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;3-Methylsulfanyl-6-[5-(4-nitro-phenyl)-furan-2-yl]-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c] cycloheptene;4-(3-Ethylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo [a,c]cyclohepten-6-yl)-benzene-1,2-diol;6-(4-Benzyloxy-phenyl)-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;6-Benzo[1,3]dioxol-5-yl-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;3-Butylsulfanyl-6-(2,4-dimethoxy-phenyl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;6-(4-Allyloxy-phenyl)-3-butylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;3-Butylsulfanyl-6-(4-ethoxy-phenyl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c] cycloheptene;6-(4-Methoxy-phenyl)-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;6-[5-(3-Nitro-phenyl)-furan-2-yl]-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene; 2-(3-Phenyl-1H-pyrazol-4-ylmethylene)-benzo[4,5]imidazo[2,1-b]thiazol-3-one;2-[5-(3-Carboxy-phenyl)-furan-2-ylmethylene]-5-(2-methoxy-naphthalen-1-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;5-(4-Dimethylamino-phenyl)-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;5-Benzo[1,3]dioxol-5-yl-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimpidine-6-carboxylicacid ethyl ester;5-(3,4-Dimethoxy-phenyl)-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;7-Methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-5-(4-methylsulfanyl-phenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;2-[5-(4-Carboxy-phenyl)-furan-2-ylmethylene]-5-(2-methoxy-naphthalen-1-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;5-Benzo[1,3]dioxol-5-yl-2-[5-(4-ethoxycarbonyl-phenyl)-furan-2-ylmethylene]-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;7-Methyl-3-oxo-5-phenyl-2-[5-(3-trifluoromethyl-phenyl)-furan-2-ylmethylene]-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester; 7-Methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-3-oxo-5-phenyl-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;2-[5-(3-Carboxy-phenyl)-furan-2-ylmethylene]-5-(4-dimethylamino-phenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;5-(4-Dimethylamino-phenyl)-7-methyl-2-[5-(4-methyl-3-nitro-phenyl)-furan-2-ylmethylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester;2-[5-(3-Carboxy-phenyl)-furan-2-ylmethylene]-7-methyl-5-(4-methylsulfanyl-phenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylicacid ethyl ester; [1,2]Naphthoquinone 1-[O-(6-oxo-6H-anthra[1,9-cd]isoxazol-5-yl)-oxime];3-Acetyl-2,5,7-triphenyl-1H-1,3a,4,8-tetraaza-7a-azonia-cyclopenta[a]indene;1-Amino-3-benzo[1,3]dioxol-5-yl-benzo[4,5]imidazo[1,2-a]pyridine-2,4-dicarbonitrile;2-[2-(5-Furan-2-yl-4-phenyl-4H-[1,2,4]triazol-3-ylsulfanyl)-acetylamino]-benzoic acid methyl ester;6,7-Dimethyl-2-(3-phenyl-1H-pyrazol-4-ylmethylene)-benzo[4,5]imidazo[2,1-b]thiazol-3-one;2-(5-Benzo[1,2,5]oxadiazol-5-yl-4-methyl-4H-[1,2,4]triazol-3-ylsulfanyl)-N-(3-methylsulfanyl-phenyl)-acetamide;4-(1,3-Dioxo-indan-2-ylidene)-2-phenyl-6-pyridin-2-yl-tetrahydro-pyrrolo[3,4-c]pyrrole-1,3-dione;6-Nitro-2-phenyl-1-(3-trifluoromethyl-benzyloxy)-1H-benzoimidazole;(6-Nitro-2-phenyl-benzoimidazol-1-yloxy)-acetic acid;1-Benzyloxy-6-nitro-2-phenyl-1H-benzoimidazole;1-(4-Methyl-benzyloxy)-6-nitro-2-phenyl-1H-benzoimidazole;6,8-Dimethyl-2-(4-nitro-phenyl)-5-phenyl-5H,6H-1-oxa-3,5,6,8-tetraaza-cyclopenta[a]naphthalene-4,7,9-trione;6,8-Dimethyl-5-phenyl-2-p-tolyl-5H,6H-1-oxa-3,5,6,8-tetraaza-cyclopenta[a]naphthalene-4,7,9-trione;2-[3-(4-Fluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl methylene]-benzo [4,5]imidazo[2,1-b]thiazol-3-one; Cobalt5,10,15,20-Tetra-pyridin-4-yl-porphyrine;2-[3-(4-Fluoro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-5-methyl-6-vinyl-imidazo[2,1-b]thiazol-3-one;Cobalt 5,10,15,20-Tetra-pyridin-3-yl-porphyrine; Zinc5,10,15,20-Tetra-pyridin-4-yl-porphyrine;2-(4-hydroxyphenyl)-4H-chromen-4-one, and pharmaceutically acceptablesalts thereof.

In a further embodiment, the transcription factor modulating compound isnot apigenin.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin). The term “aryl” also includes multicyclicaryl groups such as porphrins, phthalocyanines, etc.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 6 or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in theirring structure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkynyl groups, halogens,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term “alkylamino” includes groups and compounds wherein the nitrogen is bound to atleast one additional alkyl group. The term “dialkyl amino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “amide” or “aminocarboxy” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “electron withdrawing substituent” includes, but is not limitedto, ammonium (including alkylammonium, arylammonium, andheteroarylammonium), solfonyl Iincluding alkylsulfonyl, arylsulfonyl,and heteroarylsulfonyl), halogen, perhalogenated alkyl, cyano, oxime,carbonyl (including alkylcarbonyl, arylcarbonyl, andheteroarylcarbonyl), and nitro.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof.

Bonds represented by “{overscore (- - - - - -)}” in a structural formulamean that the bond may be either a single or a double bond.

IX. Formulations Comprising Transcription Factor Modulating Compounds

The invention provides compositions which include atherapeutically-effective amount or dose of a transcription factormodulating compound and/or a compound identified in any of the instantassays and one or more carriers (e.g., pharmaceutically acceptableadditives and/or diluents). The pharmaceutical compositions of theinvention may comprise any compound described in this application as atranscription factor modulating compound, an AraC family polypeptidemodulating compound, a MarA family polypeptide modulating compound, aMarA family inhibiting compound, a MarA inhibiting compound, compoundsof formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X),(XI), (XII), (XIII), (XIV), (XV), (XVI), (XVI), (XVIII) Table 4, Table5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11 scaffold, etc.Each of these compounds may be used alone of in combination as a part ofa pharmaceutical composition of the invention. Furthermore, acomposition can also include a second antimicrobial agent, e.g., anantibiotic.

The invention pertains to pharmaceutical compositions comprising aneffective amount of a transcription factor modulating compound (e.g., aMarA family polypeptide modulating compound or an AraC familypolypeptide modulating compound), and a pharmaceutically acceptablecarrier. In one embodiment, the transcription factor modulating compoundis of the formula (II):

wherein

W is O or S;

X is O, S, or C, optionally linked to Q;

A¹ is C-Z⁴, O, or S;

A² is C-Z⁵ or N-Z⁵;

Z¹, Z², Z³, Z⁴ and Z⁵ are each independently hydrogen, alkoxy, hydroxy,halogen, alkyl, alkenyl, alkynyl, aryl, heterocyclic, amino, or cyano;

Z³ is hydrogen, alkoxy, hydroxy, halogen, alkyl, alkenyl, alkynyl, aryl,heterocyclic, amino, nitro, cyano, carbonyl, or thiocarbonyl;

Q is an aromatic or heterocyclic moiety, and pharmaceutically acceptablesalts thereof.

In another embodiment, the pharmaceutical compositions of the inventioninclude an effective amount of a transcription factor modulatingcompound of the formula (III)

wherein

G is substituted or unsubstituted aromatic moiety, heterocyclic, alkyl,alkenyl, alkynyl, hydroxy, cyano, nitro, amino, carbonyl, or hydrogen;and

L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, and L¹⁰ are each independentlyoxygen, substituted or unsubstituted nitrogen, sulfur and or substitutedor unsubstituted carbon, and pharmaceutically acceptable salts thereof.

In yet another embodiment, the pharmaceutical compositions of theinvention include a pharmaceutically acceptable carrier (optional) andan effective amount of a transcription factor modulating compound of theformula (IV):

wherein

Y¹ and Y² are each oxygen or sulfur;

J is hydrogen, substituted or unsubstituted alkyl, alkenyl, alkynyl,cyano, nitro, amino, or halogen;

V is substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy,alkylamino, or alkylthio;

P and K are each independently substituted or unsubstituted aryl, andpharmaceutically acceptable salts thereof.

In yet another embodiment, the pharmaceutical compositions of theinvention include a pharmaceutically acceptable carrier (optional) andan effective amount of a transcription factor modulating compound of theformula (V):

wherein

T¹, T², T³, T⁴, T⁵, and T⁶ are each independently substituted orunsubstituted carbon, oxygen, substituted or unsubstituted nitrogen, orsulfur;

M is hydrogen, alkyl, alkenyl, alkynyl, or aryl, or pharmaceuticallyacceptable salts thereof.

In yet another embodiment, the pharmaceutical compositions of theinvention include a pharmaceutically acceptable carrier (optional) andan effective amount of a transcription factor modulating compound of theformula (Va):

wherein

R¹ is OH, OCOCO₂H, or a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group;

R² is H, CO₂(C₁-C₅ substituted or unsubstituted, straight or branchedalkyl), or a substituted or unsubstituted aryl group; and

R⁴, R⁵, R⁶, and R⁷ are independently selected from the group consistingof H, (C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO₂(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(C₁-C₅ substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen.

In certain embodiments of formula Va, those compounds disclosed in U.S.Ser. No. 10/139,591, filed May 6, 2002, are excluded from the scope ofthe present invention.

In other embodiments of formula Va, when R⁶ is NO₂ and R² isunsubstituted phenyl, then R¹ is not O(CHCH₃)(CO₂)CH₂CH₃ or OCH₂CO₂H.Also, in another embodiment, when R⁶ is H or NO₂, then R¹ is not aphenyl-substituted alkyloxy group. In yet another embodiment, when R⁴,R⁵, R⁶, and R⁷ are all H and R² is para-methoxyphenyl, then R¹ is notOH. And in another embodiment, when R⁴, R⁵, R⁶, and R⁷ are all H and R²is unsubstituted phenyl, then R¹ is not OCH₂CO₂CH₂CH₃;

In certain aspects of formula Va, R⁴, R⁵, and R⁷ are all H.

Similarly, R¹ of formula Va may be selected from the group consisting ofOH, O(CR′R″)₁₋₃H, O(CR′R″)₁₋₃OH, O(CR′R″)₁₋₃CO₂H,O(CR′R″)₁₋₃CO₂(CR′R″)₁₋₃H, O(CR′R″)₁₋₃(CO)NH₂, O(CR′R″)₁₋₃(CNH)NH₂,OCOCO₂H, O(CR′R″)₁₋₃SO₃H, O(CR′R″)₁₋₃OSO₃H, O(CR′R″)₁₋₃PO₃H,O(CR′R″)₁₋₃OPO₃H, O(CR′R″)₁₋₃N[(CR′R″)₀₋₃H]₂, O(CR′R″)₁₋₃(CO)(NHOH), andO(CR′R″)₁₋₃(heteroaryl); wherein R′ and R″ are each independently H, aC₁-C₃ alkyl, C₂-C₃ alkenyl, or C₂-C₃ alkynyl group. Each R′ and R″ ispreferably H or CH₃.

When R¹ of formula Va is O(CR′R″)₁₋₃(heteroaryl), the heteroaryl groupmay be a pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl,imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl,pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.

Similarly, when R² of formula Va may be a substituted or unsubstitutedphenyl, pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiaozolyl,imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl,pyridinyl, pyrazinyl, pyridazinyl, or pyrimidinyl group.

In a more particular embodiment, R⁶ of formula Va is H, (CR′R″)₁₋₃H,(CR′R″)₁₋₃OH, (CR′R″)₁₋₃NH₂, (NOH)(CR′R″)₁₋₃H, CO(CR′R″)₀₋₃NH₂,CO(CR′R″)₁₋₃H, CO(CR′R″)₁₋₃OH, CO(CR′R″)₀₋₃CF₃,(CR′R″)₀₋₃N[(CR′R″)₀₋₃H]₂, CO(substituted or unsubstituted heteroaryl),CO(C₃-C₆ substituted or unsubstituted cycloalkyl), O(CR′R″)₁₋₃H,CO(substituted or unsubstituted phenyl), CO₂(CR′R″)₀₋₃H, CN, NO₂, F, Cl,Br, or I, wherein R′ and R″ are each independently H, a C₁-C₃ alkyl,C₂-C₃ alkenyl, or C₂-C₃ alkynyl group. Preferably each R′ and R″ isindependently H or CH₃.

In yet another embodiment, R⁶ of formula Va is CO(substituted orunsubstituted heteroaryl), wherein said heteroaryl group is a pyrrolyl,furanyl, thiophenyl, thiazolyl, isothiaozolyl, imidazolyl, triazolyl,tetrazolyl, pyrazolyl, oxazolyl, isooxazolyl, pyridinyl, pyrazinyl,pyridazinyl, or pyrimidinyl group.

In still another embodiment of formula Va, R¹ is OH, OCOCO₂H, or asubstituted straight or branched C₁-C₅ alkyloxy group, provided that R¹is not a 2-amino-substituted ethoxy group or a substituted orunsubstituted benzyloxy group; R² is H, CO₂(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), or a substituted orunsubstituted aryl group, provided that said aryl group is not athiazolyl or isothiazolyl group; and R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of H, (C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO₂(C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO(C₁-C₅substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; provided that whenR⁶ is NO₂ and R² is unsubstituted phenyl, then R¹ is notO(CHCH₃)(CO₂)CH₂CH₃ or OCH₂CO₂H; provided that when R⁶ is H or NO₂, thenR¹ is not a phenyl-substituted alkyloxy group; provided that when R⁴,R⁵, R⁶, and R⁷ are all H and R² is para-methoxyphenyl, then R¹ is notOH; and provided that when R⁴, R⁵, R⁶, and R⁷ are all H and R² isunsubstituted phenyl, or when R⁴, R⁵, and R⁷ are all H, R⁶ is Cl, and R²is para-methyl-phenyl, then R¹ is not OCH₂CO₂CH₂CH₃

In another embodiment, R⁶ of formula Va is an electron withdrawingsubstituent, selected from the group consisting of F, CF₃, NO₂,C(NOH)(CR′R″), wherein each R′ and R″ are each independently H or CH₃.

In yet another embodiment, the pharmaceutical compositions of theinvention include a pharmaceutically acceptable carrier (optional) andan effective amount of a transcription factor modulating compound of theformula (VI):

wherein

G¹, G², and G³ are each independently O, S, substituted or unsubstitutednitrogen, or substituted or unsubstituted carbon;

E¹, E², and E³ are each independently hydrogen, alkyl, alkenyl, alkynyl,aryl, aralkyl, or acyl; and

E⁴ is alkyl, alkenyl, alkynyl, aryl, halogen, cyano, amino, nitro, oracyl, and pharmaceutically acceptable salts thereof.

In yet another further embodiment, the pharmaceutical compositions ofthe invention comprise an effective amount of a transcription factormodulating compound listed below or found in Table 4, Table 5, Table 6,Table 7, Table 8, Table 9, Table 10, Table 11:

-   2-(4-isopropylphenyl)-4H-chromen-4-one;    2-(3,4-Dihydroxy-phenyl)-3,5,7-trihydroxy-chromen-4-one,    N-isopropyl-2-[(4-methyl-5-quinolin-6-yl-4H-1,2,4-triazol-3-yl)thio]acetamide;    4-hydroxy-6-methyl-5,6-dihydro-2H-pyrano[3,2-c]quinoline-2,5-dione;    5,7-Dihydroxy-2-(4-hydroxy-phenyl)-chromen-4-one;    2-[4-(dimethylamino)phenyl]-4H-chromen-4-one;    1-(benzyloxy)-2-phenyl-1H-imidazo[4,5-b]pyridine;    2-(benzylthio)-4-phenyl-5-(1-phenyl-1H-1,2,3,4-tetraazol-5-yl)pyrimidine;    6-fluoro-2-phenyl-4H-chromen-4-one;    7-methoxy-2-phenyl-4H-chromen-4-one;    4-(1,3-dioxo-1,3-dihydro-2H-inden-2-yliden)-2-phenyl-6-(2-pyridinyl)tetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione;    2-(2-Hydroxy-3-oxo-5-p-tolyl-2,3-dihydro-furan-2-yl)-malonamic acid    ethyl ester;    2-[(6-nitro-2-phenyl-1H-1,3-benzimidazol-1-yl)oxy]acetic acid;    2-(4-fluorophenyl)-4H-chromen-4-one;    1-methoxy-2-(4-methylphenyl)-1H-imidazo[4,5-b]pyridine;    6-(5-Iodo-furan-2-yl)-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c]cycloheptene;    6-(4-Ethoxy-phenyl)-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c]cycloheptene;    3-Methylsulfanyl-6-(5-nitro-furan-2-yl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c]cycloheptene;    3-Methylsulfanyl-6-[5-(4-nitro-phenyl)-furan-2-yl]-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c] cycloheptene;    4-(3-Ethylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo [a,c]    cyclohepten-6-yl)-benzene-1,2-diol;    6-(4-Benzyloxy-phenyl)-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c]cycloheptene;    6-Benzo[1,3]dioxol-5-yl-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c]cycloheptene;    3-Butylsulfanyl-6-(2,4-dimethoxy-phenyl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]    cycloheptene;    6-(4-Allyloxy-phenyl)-3-butylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene;    3-Butylsulfanyl-6-(4-ethoxy-phenyl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c] cycloheptene;    6-(4-Methoxy-phenyl)-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c]cycloheptene;    6-[5-(3-Nitro-phenyl)-furan-2-yl]-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo    [a,c] cycloheptene; 2-(3-Phenyl-1H-pyrazol-4-ylmethylene)-benzo[4,5]    imidazo[2,1-b]thiazol-3-one;    2-[5-(3-Carboxy-phenyl)-furan-2-ylmethylene]-5-(2-methoxy-naphthalen-1-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    5-(4-Dimethylamino-phenyl)-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl    methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    5-Benzo[1,3]dioxol-5-yl-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl    methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    5-(3,4-Dimethoxy-phenyl)-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl    methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    7-Methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-5-(4-methyl    sulfanyl-phenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    2-[5-(4-Carboxy-phenyl)-furan-2-ylmethylene]-5-(2-methoxy-naphthalen-1-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    5-Benzo[1,3]dioxol-5-yl-2-[5-(4-ethoxycarbonyl-phenyl)-furan-2-ylmethylene]-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    7-Methyl-3-oxo-5-phenyl-2-[5-(3-trifluoromethyl-phenyl)-furan-2-ylmethylene]-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester; 7-Methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl    methylene]-3-oxo-5-phenyl-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    2-[5-(3-Carboxy-phenyl)-furan-2-ylmethylene]-5-(4-dimethylamino-phenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    5-(4-Dimethylamino-phenyl)-7-methyl-2-[5-(4-methyl-3-nitro-phenyl)-furan-2-yl    methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester;    2-[5-(3-Carboxy-phenyl)-furan-2-ylmethylene]-7-methyl-5-(4-methylsulfanyl-phenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic    acid ethyl ester; [1,2]Naphthoquinone 1-[O-(6-oxo-6H-anthra[1,9-cd]    isoxazol-5-yl)-oxime];    3-Acetyl-2,5,7-triphenyl-1H-1,3a,4,8-tetraaza-7a-azonia-cyclopenta[a]indene;    1-Amino-3-benzo[1,3]dioxol-5-yl-benzo[4,5]imidazo[1,2-a]pyridine-2,4-dicarbonitrile;    2-[2-(5-Furan-2-yl-4-phenyl-4H-[1,2,4]triazol-3-yl    sulfanyl)-acetylamino]-benzoic acid methyl ester;    6,7-Dimethyl-2-(3-phenyl-1H-pyrazol-4-ylmethylene)-benzo[4,5]imidazo[2,1-b]thiazol-3-one;    2-(5-Benzo[1,2,5]oxadiazol-5-yl-4-methyl-4H-[1,2,4]    triazol-3-ylsulfanyl)-N-(3-methylsulfanyl-phenyl)-acetamide;    4-(1,3-Dioxo-indan-2-ylidene)-2-phenyl-6-pyridin-2-yl-tetrahydro-pyrrolo[3,4-c]pyrrole-1,3-dione;    6-Nitro-2-phenyl-1-(3-trifluoromethyl-benzyloxy)-1H-benzoimidazole;    (6-Nitro-2-phenyl-benzoimidazol-1-yloxy)-acetic acid;    1-Benzyloxy-6-nitro-2-phenyl-1H-benzoimidazole;    1-(4-Methyl-benzyloxy)-6-nitro-2-phenyl-1H-benzoimidazole;    6,8-Dimethyl-2-(4-nitro-phenyl)-5-phenyl-5H,    6H-1-oxa-3,5,6,8-tetraaza-cyclopenta[a]naphthalene-4,7,9-trione;    6,8-Dimethyl-5-phenyl-2-p-tolyl-5H,6H-1-oxa-3,5,6,8-tetraaza-cyclopenta    [a]naphthalene-4,7,9-trione;    2-[3-(4-Fluoro-phenyl)-1-phenyl-1H-pyrazol-4-yl methylene]-benzo    [4,5] imidazo[2,1-b]thiazol-3-one; Cobalt    5,10,15,20-Tetra-pyridin-4-yl-porphyrine;    2-[3-(4-Fluoro-phenyl)-1-phenyl-1H-pyrazol-4-ylmethylene]-5-methyl-6-vinyl-imidazo[2,1-b]thiazol-3-one;    Cobalt 5,10,15,20-Tetra-pyridin-3-yl-porphyrine; Zinc    5,10,15,20-Tetra-pyridin-4-yl-porphyrine;    2-(4-hydroxyphenyl)-4H-chromen-4-one, and pharmaceutically    acceptable salts thereof.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atranscription factor modulating compound, wherein said compound is ofthe formula (XI), (XII), (XIII), (XIV), (XV), (XVI), or (XVII). Inanother embodiment, the pharmaceutical composition can further comprisean antibiotic. In a further embodiment, the effective amount of thepharmaceutical composition can be effective for treating a biofilmassociated state in a subject. The biofilm associated states caninclude, for example, middle ear infections, cystic fibrosis,osteomyelitis, acne, dental cavities, endocarditis, and prostatitis.

In another embodiment, the method for preventing a bacterial associatedstate in a subject, comprising administering to the subject an effectiveamount of a transcription factor modulating compound, such that thebacterial associated state is prevented. In a further embodiment, thetranscription factor modulating compound is of the formula (XI), (XII),(XIII), (XIV), (XV), (XVI), or (XVII). In a further embodiment, thetranscription factor modulating compound can include, for example, aMarA family polypeptide inhibitor and an AraC family polypeptideinhibitor.

The term “subject” includes plants and animals (e.g., vertebrates,amphibians, fish, mammals, e.g., cats, dogs, horses, pigs, cows, sheep,rodents, rabbits, squirrels, bears, primates (e.g., chimpanzees,gorillas, and humans) which are capable of suffering from a bacterialassociated disorder. The term “subject” also comprises immunocompromisedsubjects, who may be at a higher risk for infection.

The term “preventing” the administration of an effective amount of thetranscription factor modulating compound to prevent a bacterialassociated state from occurring.

The term “bacterial associated state” includes states characterized bythe presence of bacteria which can be prevented by administering thetranscription factor modulating compounds of the invention. The termincludes biofilm associated states and other infections or theundesirable presence of a bacteria on or in a subject.

As described in detail below, the pharmaceutical compositions can beformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example, aqueousor non-aqueous solutions or suspensions, tablets, boluses, powders,granules, pastes; (2) parental administration, for example, bysubcutaneous, intramuscular or intravenous injection as, for example, asterile solution or suspension; (3) topical application, for example, asa cream, ointment or spray applied to the skin; (4) intravaginally orintrarectally, for example, as a pessary, cream, foam, or suppository;or (5) aerosol, for example, as an aqueous aerosol, liposomalpreparation or solid particles containing the compound.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the antiinfective agentsor compounds of the invention from one organ, or portion of the body, toanother organ, or portion of the body without affecting its biologicaleffect. Each carrier should be “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the subject. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalcompositions. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microbes may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Pharmaceutical compositions of the present invention may be administeredto epithelial surfaces of the body orally, parenterally, topically,rectally, nasally, intravaginally, intracisternally. They are of coursegiven by forms suitable for each administration route. For example, theyare administered in tablets or capsule form, by injection, inhalation,eye lotion, ointment, etc., administration by injection, infusion orinhalation; topical by lotion or ointment; and rectal or vaginalsuppositories.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a sucrose octasulfate and/or anantibacterial, drug or other material other than directly into thecentral nervous system, such that it enters the subject's system and,thus, is subject to metabolism and other like processes, for example,subcutaneous administration.

In some methods, the compositions of the invention can be topicallyadministered to any epithelial surface. An “epithelial surface”according to this invention is defined as an area of tissue that coversexternal surfaces of a body, or which lines hollow structures including,but not limited to, cutaneous and mucosal surfaces. Such epithelialsurfaces include oral, pharyngeal, esophageal, pulmonary, ocular, aural,nasal, buccal, lingual, vaginal, cervical, genitourinary, alimentary,and anorectal surfaces.

Compositions can be formulated in a variety of conventional formsemployed for topical administration. These include, for example,semi-solid and liquid dosage forms, such as liquid solutions orsuspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions, slurries, powders, sprays, lipsticks, foams, pastes,toothpastes, ointments, salves, balms, douches, drops, troches, chewinggums, lozenges, mouthwashes, rinses.

Conventionally used carriers for topical applications include pectin,gelatin and derivatives thereof, polylactic acid or polyglycolic acidpolymers or copolymers thereof, cellulose derivatives such as methylcellulose, carboxymethyl cellulose, or oxidized cellulose, guar gum,acacia gum, karaya gum, tragacanth gum, bentonite, agar, carbomer,bladderwrack, ceratonia, dextran and derivatives thereof, ghatti gum,hectorite, ispaghula husk, polyvinypyrrolidone, silica and derivativesthereof, xanthan gum, kaolin, talc, starch and derivatives thereof,paraffin, water, vegetable and animal oils, polyethylene, polyethyleneoxide, polyethylene glycol, polypropylene glycol, glycerol, ethanol,propanol, propylene glycol (glycols, alcohols), fixed oils, sodium,potassium, aluminum, magnesium or calcium salts (such as chloride,carbonate, bicarbonate, citrate, gluconate, lactate, acetate, gluceptateor tartrate).

Such compositions can be particularly useful, for example, for treatmentor prevention of an unwanted cell, e.g., vaginal Neisseria gonorrhoeae,or infections of the oral cavity, including cold sores, infections ofeye, the skin, or the lower intestinal tract. Standard compositionstrategies for topical agents can be applied to the antiinfectivecompounds or a pharmaceutically acceptable salt thereof in order toenhance the persistence and residence time of the drug, and to improvethe prophylactic efficacy achieved.

For topical application to be used in the lower intestinal tract orvaginally, a rectal suppository, a suitable enema, a gel, an ointment, asolution, a suspension or an insert can be used. Topical transdermalpatches may also be used. Transdermal patches have the added advantageof providing controlled delivery of the compositions of the invention tothe body. Such dosage forms can be made by dissolving or dispersing theagent in the proper medium.

Compositions of the invention can be administered in the form ofsuppositories for rectal or vaginal administration. These can beprepared by mixing the agent with a suitable non-irritating carrierwhich is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum or vagina to release the drug. Suchmaterials include cocoa butter, beeswax, polyethylene glycols, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Compositions which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, films, or spraycompositions containing such carriers as are known in the art to beappropriate. The carrier employed in the sucroseoctasulfate/contraceptive agent should be compatible with vaginaladministration and/or coating of contraceptive devices. Combinations canbe in solid, semi-solid and liquid dosage forms, such as diaphragm,jelly, douches, foams, films, ointments, creams, balms, gels, salves,pastes, slurries, vaginal suppositories, sexual lubricants, and coatingsfor devices, such as condoms, contraceptive sponges, cervical caps anddiaphragms.

For ophthalmic applications, the pharmaceutical compositions can beformulated as micronized suspensions in isotonic, pH adjusted sterilesaline, or, preferably, as solutions in isotonic, pH adjusted sterilesaline, either with or without a preservative such as benzylalkoniumchloride. Alternatively, for ophthalmic uses, the compositions can beformulated in an ointment such as petrolatum. Exemplary ophthalmiccompositions include eye ointments, powders, solutions and the like.

Powders and sprays can contain, in addition to sucrose octasulfateand/or antibiotic or contraceptive agent(s), carriers such as lactose,talc, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), proteins like serum albumin, sorbitan esters,oleic acid, lecithin, amino acids such as glycine, buffers, salts,sugars or sugar alcohols. Aerosols generally are prepared from isotonicsolutions.

Compositions of the invention can also be orally administered in anyorally-acceptable dosage form including, but not limited to, capsules,cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), powders, granules, or as a solutionor a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of sucrose octasulfate and/or antibiotic orcontraceptive agent(s) as an active ingredient. A compound may also beadministered as a bolus, electuary or paste. In the case of tablets fororal use, carriers which are commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may be scored or prepared with coatings and shells, suchas enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the antiinfective agent(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Sterile injectable forms of the compositions of this invention can beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. Wetting agents, emulsifiers andlubricants, such as sodium lauryl sulfate and magnesium stearate, aswell as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a nontoxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as Ph. Helv or similar alcohol.

The antiinfective agent or a pharmaceutically acceptable salt thereofwill represent some percentage of the total dose in other dosage formsin a material forming a combination product, including liquid solutionsor suspensions, suppositories, douches, enemas, gels, creams, emulsions,lotions slurries, soaps, shampoos, detergents, powders, sprays,lipsticks, foams, pastes, toothpastes, ointments, salves, balms,douches, drops, troches, lozenges, mouthwashes, rinses and others.Creams and gels for example, are typically limited by the physicalchemical properties of the delivery medium to concentrations less than20% (e.g., 200 mg/gm). For special uses, far less concentratedpreparations can be prepared, (e.g., lower percent formulations forpediatric applications). For example, the pharmaceutical composition ofthe invention can comprise sucrose octasulfate in an amount of0.001-99%, typically 0.01-75%, more typically 0.1-20%, especially 1-10%by weight of the total preparation. In particular, a preferredconcentration thereof in the preparation is 0.5-50%, especially 0.5-25%,such as 1-10%. It can be suitably applied 1-10 times a day, depending onthe type and severity of the condition to be treated or prevented.

Given the low toxicity of an antiinfective agent or a pharmaceuticallyacceptable salt thereof over many decades of clinical use as ananti-ulcerant [W. R. Garnett, Clin. Pharm. 1:307-314 (1982); R. N.Brogden et al., Drugs 27:194-209 (1984); D. M. McCarthy, New Eng J Med.,325:1017-1025 (1991), an upper limit for the therapeutically effectivedose is not a critical issue.

For prophylactic applications, the pharmaceutical composition of theinvention can be applied prior to potential infection. The timing ofapplication prior to potential infection can be optimized to maximizethe prophylactic effectiveness of the compound. The timing ofapplication will vary depending on the mode of administration, theepithelial surface to which it is applied, the surface area, doses, thestability and effectiveness of composition under the pH of theepithelial surface, the frequency of application, e.g., singleapplication or multiple applications. One skilled in the art will beable to determine the most appropriate time interval required tomaximize prophylactic effectiveness of the compound.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, genetics, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Genetics; MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al. (ColdSpring Harbor Laboratory Press (1989)); Short Protocols in MolecularBiology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY (1995)); DNACloning, Volumes I and II (D. N. Glover ed., 1985); OligonucleotideSynthesis (M. J. Gait ed. (1984)); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1984)); the treatise, Methods In Enzymology (Academic Press, Inc.,N.Y); Immunochemical Methods In Cell And Molecular Biology (Mayer andWalker, eds., Academic Press, London (1987)); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. (1986));and Miller, J. Experiments in Molecular Genetics (Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1972)).

X. The Role of Transcription Activation Factor Polypeptides in Biofilms

In one embodiment, the invention pertains to a method for dispersing orpreventing the formation of a biofilm on a surface or in an area, byadministering an effective amount of a transcription factor modulatingcompound, e.g., a HTH protein modulating compound, an AraC familypolypeptide modulating compound, a MarA family polypeptide modulatingcompound, or a MarA inhibiting compound.

It has been discovered that the absence of MarA and its homologs has anegative effect on biofilm formation in E. coli. In order to confirmthis finding genetically, plasmid encoded marA was transformed into anE. coli strain deleted of marA, soxS, and rob (triple knockout). Theexpression of MarA in this triple knockout restored biofilm formation inthis host to a level that was comparable to that of the wild type host.

The term “biofilm” includes biological films that develop and persist atinterfaces in aqueous and other environments. Biofilms are composed ofmicroorganisms embedded in an organic gelatinous structure composed ofone or more matrix polymers which are secreted by the residentmicroorganisms. The term “biofilm” also includes bacteria that areattached to a surface in sufficient numbers to be detected orcommunities of microorganisms attached to a surface (Costerton, J. W.,et al. (1987) Ann. Rev. Microbiol. 41:435-464; Shapiro, J. A. (1988) SciAm. 256:82-89; O'Toole, G. et al. (2000) Annu Rev Microbiol. 54:49-79).

In another embodiment, the invention pertains to methods of treatingbiofilm associated states in a subject, by administering to said subjectan effective amount of a transcription factor modulating compound, e.g.,a MarA family inhibiting compound, such that the biofilm associatedstate is treated.

The term “biofilm associated states” includes disorders which arecharacterized by the presence or potential presence of a bacterialbiofilm. Many medically important pathogens form biofilms and biofilmformation is often one component of the infectious process (Costerton,J. W. et al. (1999) Science 284:1318-1322). Examples of biofilmassociated states include, but are not limited to, middle earinfections, cystic fibrosis, osteomyelitis, acne, dental cavities, andprostatitis. Biofilm associated states also include infection of thesubject by one or more bacteria, e.g., Pseudomonas aeruginosa. Oneconsequence of biofilm formation is that bacteria within biofilms aregenerally less susceptible to a range of different antibiotics relativeto their planktonic counterparts.

Furthermore, the invention also pertains to methods for preventing theformation of biofilms on surfaces or in areas, by contacting the areawith an effective amount of a transcription factor modulating compound,e.g., a MarA family inhibiting compound, etc.

Industrial facilities employ many methods of preventing biofouling ofindustrial water systems. Many microbial organisms are involved inbiofilm formation in industrial waters. Growth of slime-producingbacteria in industrial water systems causes problems including decreasedheat transfer, fouling and blockage of lines and valves, and corrosionor degradation of surfaces. Control of bacterial growth in the past hasbeen accomplished with biocides. Many biocides and biocide formulationsare known in the art. However, many of these contain components whichmay be environmentally deleterious or toxic, and are often resistant tobreakdown.

The transcription factor inhibiting compounds, such as but not limitedto AraC family inhibiting compounds and MarA family inhibitingcompounds, of the present invention are useful in a variety ofenvironments including industrial, clinical, the household, and personalcare. The compositions of the invention may comprise one or morecompounds of the invention as an active ingredient acting alone,additively, or synergistically against the target organism.

The MarA family inhibiting compounds and modulating compounds of theinvention may be formulated in a composition suitable for use inenvironments including industry, pharmaceutics, household, and personalcare. In an embodiment, the compounds of the invention are soluble inwater. The modulating compounds may be applied or delivered with anacceptable carrier system. The composition may be applied or deliveredwith a suitable carrier system such that the active ingredient (e.g.,transcription factor modulating compound of the invention such as a MarAfamily modulating compound, e.g., a MarA family polypeptide inhibitingcompound) may be dispersed or dissolved in a stable manner so that theactive ingredient, when it is administered directly or indirectly, ispresent in a form in which it is available in a advantageous way.

Also, the separate components of the compositions of the invention maybe preblended or each component may be added separately to the sameenvironment according to a predetermined dosage for the purpose ofachieving the desired concentration level of the treatment componentsand so long as the components eventually come into intimate admixturewith each other. Further, the present invention may be administered ordelivered on a continuous or intermittent basis.

A transcription factor modulating compound, e.g., a MarA familymodulating compound of the present invention, when present in acomposition will generally be present in an amount from about 0.000001%to about 100%, more preferably from about 0.001% to about 50%, and mostpreferably from about 0.01% to about 25%.

For compositions of the present invention comprising a carrier, thecomposition comprises, for example, from about 1% to about 99%,preferably from about 50% to about 99%, and most preferably from about75% to about 99% by weight of at least one carrier.

The transcription factor modulating compound, e.g., the MarA familypolypeptide inhibiting compound, of the invention may be formulated withany suitable carrier and prepared for delivery in forms, such as,solutions, microemulsions, suspensions or aerosols. Generation of theaerosol or any other means of delivery of the present invention may beaccomplished by any of the methods known in the art. For example, in thecase of aerosol delivery, the compound is supplied in a finely dividedform along with any suitable carrier with a propellant. Liquefiedpropellants are typically gases at ambient conditions and are condensedunder pressure. The propellant may be any acceptable and known in theart including propane and butane, or other lower alkanes, such as thoseof up to 5 carbons. The composition is held within a container with anappropriate propellant and valve, and maintained at elevated pressureuntil released by action of the valve.

The compositions of the invention may be prepared in a conventional formsuitable for, but not limited to topical or local application such as anointment, paste, gel, spray and liquid, by including stabilizers,penetrants and the carrier or diluent with the compound according to aknown technique in the art. These preparations may be prepared in aconventional form suitable for enteral, parenteral, topical orinhalational applications.

The present invention may be used in compositions suitable for householduse. For example, compounds of the present invention are also useful asactive antimicrobial ingredients in household products such ascleansers, detergents, disinfectants, dishwashing liquids, soaps anddetergents. In an embodiment, the transcription factor modulatingcompound of the present invention may be delivered in an amount and formeffective for the prevention, removal or termination of microbes.

The compositions of the invention for household use comprise, forexample, at least one transcription factor modulating compound of theinvention and at least one suitable carrier. For example, thecomposition may comprise from about 0.00001% to about 50%, preferablyfrom about 0.0001% to about 25%, most preferably from about 0.0005% toabout 10% by weight of the modulating compound based on the weightpercentage of the total composition.

The transcription factor modulating compound of the present inventionmay also be used in hygiene compositions for personal care. Forinstance, compounds of the invention can be used as an active ingredientin personal care products such as facial cleansers, astringents, bodywash, shampoos, conditioners, cosmetics and other hygiene products. Thehygiene composition may comprise any carrier or vehicle known in the artto obtain the desired form (such as solid, liquid, semisolid or aerosol)as long as the effects of the compound of the present invention are notimpaired. Methods of preparation of hygiene compositions are notdescribed herein in detail, but are known in the art. For its discussionof such methods, The CTFA Cosmetic Ingredient Handbook, Second Edition,1992, and pages 5-484 of A Formulary of Cosmetic Preparations (Vol. 2,Chapters 7-16) are incorporated herein by reference.

The hygiene composition for use in personal care comprise generally atleast one modulating compound of the present application and at leastone suitable carrier. For example, the composition may comprise fromabout 0.00001% to about 50%, preferably from about 0.0001% to about 25%,more preferably from about 0.0005% to about 10% by weight of thetranscription factor modulating compound of the invention based on theweight percentage of the total composition.

The transcription factor modulating compound of the present inventionmay be used in industry. In the industrial setting, the presence ofmicrobes can be problematic, as microbes are often responsible forindustrial contamination and biofouling. Compositions of the inventionfor industrial applications may comprise an effective amount of thecompound of the present invention in a composition for industrial usewith at least one acceptable carrier or vehicle known in the art to beuseful in the treatment of such systems. Such carriers or vehicles mayinclude diluents, deflocculating agents, penetrants, spreading agents,surfactants, suspending agents, wetting agents, stabilizing agents,compatibility agents, sticking agents, waxes, oils, co-solvents,coupling agents, foams, antifoaming agents, natural or syntheticpolymers, elastomers and synergists. Methods of preparation, deliverysystems and carriers for such compositions are not described here indetail, but are known in the art. For its discussion of such methods,U.S. Pat. No. 5,939,086 is herein incorporated by reference.Furthermore, the preferred amount of the composition to be used may varyaccording to the active ingredient(s) and situation in which thecomposition is being applied.

The transcription factor modulating compounds, e.g., MarA familypolypeptide inhibiting compounds, and compositions of the presentinvention may be useful in nonaqueous environments. Such nonaqueousenvironments may include, but are not limited to, terrestrialenvironments, dry surfaces or semi-dry surfaces in which the compound orcomposition is applied in a manner and amount suitable for thesituation.

The transcription factor modulating compounds, e.g., MarA familypolypeptide modulating compounds, e.g., MarA inhibiting compounds, ofthe present invention may be used to form contact-killing coatings orlayers on a variety of substrates including personal care products (suchas toothbrushes, contact lens cases and dental equipment), healthcareproducts, household products, food preparation surfaces and packaging,and laboratory and scientific equipment. Further, other substratesinclude medical devices such as catheters, urological devices, bloodcollection and transfer devices, tracheotomy devices, intraocularlenses, wound dressings, sutures, surgical staples, membranes, shunts,gloves, tissue patches, prosthetic devices (e.g., heart valves) andwound drainage tubes. Still further, other substrates include textileproducts such as carpets and fabrics, paints and joint cement. A furtheruse is as an antimicrobial soil fumigant.

The transcription factor modulating compounds of the invention may alsobe incorporated into polymers, such as polysaccharides (cellulose,cellulose derivatives, starch, pectins, alginate, chitin, guar,carrageenan), glycol polymers, polyesters, polyurethanes, polyacrylates,polyacrylonitrile, polyamides (e.g., nylons), polyolefins, polystyrenes,vinyl polymers, polypropylene, silks or biopolymers. The modulatingcompounds may be conjugated to any polymeric material such as those withthe following specified functionality: 1) carboxy acid, 2) amino group,3) hydroxyl group and/or 4) haloalkyl group.

The composition for treatment of nonaqueous environments may be compriseat least one transcription factor modulating compound of the presentapplication and at least one suitable carrier. In an embodiment, thecomposition comprises from about 0.001% to about 75%, advantageouslyfrom about 0.01% to about 50%, and preferably from about 0.1% to about25% by weight of a transcription factor modulating compound of theinvention based on the weight percentage of the total composition.

The transcription factor modulating compounds and compositions of theinvention may also be useful in aqueous environments. “Aqueousenvironments” include any type of system containing water, including,but not limited to, natural bodies of water such as lakes or ponds;artificial, recreational bodies of water such as swimming pools and hottubs; and drinking reservoirs such as wells. The compositions of thepresent invention may be useful in treating microbial growth in theseaqueous environments and may be applied, for example, at or near thesurface of water.

The compositions of the invention for treatment of aqueous environmentsmay comprise at least one transcription factor modulating compound ofthe present invention and at least one suitable carrier. In anembodiment, the composition comprises from about 0.001% to about 50%,advantageously from about 0.003% to about 15%, preferably from about0.01% to about 5% by weight of the compound of the invention based onthe weight percentage of the total composition.

The present invention also provides a process for the production of anantibiofouling composition for industrial use. Such process comprisesbringing at least one of any industrially acceptable carrier known inthe art into intimate admixture with a transcription factor modulatingcompound of the present invention. The carrier may be any suitablecarrier discussed above or known in the art.

The suitable antibiofouling compositions may be in any acceptable formfor delivery of the composition to a site potentially having, or havingat least one living microbe. The antibiofouling compositions may bedelivered with at least one suitably selected carrier as hereinbeforediscussed using standard formulations. The mode of delivery may be suchas to have a binding inhibiting effective amount of the antibiofoulingcomposition at a site potentially having, or having at least one livingmicrobe. The antibiofouling compositions of the present invention areuseful in treating microbial growth that contributes to biofouling, suchas scum or slime formation, in these aqueous environments. Examples ofindustrial processes in which these compounds might be effective includecooling water systems, reverse osmosis membranes, pulp and papersystems, air washer systems and the food processing industry. Theantibiofouling composition may be delivered in an amount and formeffective for the prevention, removal or termination of microbes.

The antibiofouling composition of the present invention generallycomprise at least one compound of the invention. The composition maycomprise from about 0.001% to about 50%, more preferably from about0.003% to about 15%, most preferably from about 0.01% to about 5% byweight of the compound of the invention based on the weight percentageof the total composition.

The amount of antibiofouling composition may be delivered in an amountof about 1 mg/l to about 1000 mg/l, advantageously from about 2 mg/l toabout 500 mg/l, and preferably from about 20 mg/l to about 140 mg/l.

Antibiofouling compositions for water treatment generally comprisetranscription factor modulating compounds of the invention in amountsfrom about 0.001% to about 50% by weight of the total composition. Othercomponents in the antibiofouling compositions (used at 0.1% to 50%) mayinclude, for example, 2-bromo-2-nitropropane-1,3-diol (BNPD),β-nitrostyrene (BNS), dodecylguanidine hydrochloride,2,2-dibromo-3-nitrilopropionamide (DBNPA), glutaraldehyde, isothiazolin,methylene bis(thiocyanate), triazines, n-alkyl dimethylbenzylammoniumchloride, trisodium phosphate-based, antimicrobials, tributyltin oxide,oxazolidines, tetrakis (hydroxymethyl)phosphonium sulfate (THPS),phenols, chromated copper arsenate, zinc or copper pyrithione,carbamates, sodium or calcium hypochlorite, sodium bromide,halohydantoins (Br, Cl), or mixtures thereof.

Other possible components in the compositions of the invention includebiodispersants (about 0.1% to about 15% by weight of the totalcomposition), water, glycols (about 20-30%) or Pluronic (atapproximately 7% by weight of the total composition). The concentrationof antibiofouling composition for continuous or semi-continuous use isabout 5 to about 70 mg/l.

Antibiofouling compositions for industrial water treatment may comprisecompounds of the invention in amounts from about 0.001% to about 50%based on the weight of the total composition. The amount of compound ofthe invention in antibiofouling compositions for aqueous water treatmentmay be adjusted depending on the particular environment. Shock doseranges are generally about 20 to about 140 mg/l; the concentration forsemi-continuous use is about 0.5× of these concentrations.

The invention also pertains, at least in part, to a method of regulatingbiofilm development. The method includes administering a compositionwhich contains a transcription factor modulating compound of theinvention. The composition can also include other components whichenhance the ability of the composition to degrade biofilms.

The composition can be formulated as a cleaning product, e.g., ahousehold or an industrial cleaner to remove, prevent, inhibit, ormodulate biofilm development. Advantageously, the biofilm is adverselyaffected by the administration of the compound of the invention, e.g.,biofilm development is diminished. These compositions may includecompounds such as disinfectants, soaps, detergents, as well as othersurfactants. Examples of surfactants include, for example, sodiumdodecyl sulfate; quaternary ammonium compounds; alkyl pyridiniumiodides; TWEEN 80, TWEEN 85, TRITON X-100; BRIJ 56; biologicalsurfactants; rhamnolipid, surfactin, visconsin, and sulfonates. Thecomposition of the invention may be applied in known areas and surfaceswhere disinfection is required, including but not limited to drains,shower curtains, grout, toilets and flooring. A particular applicationis on hospital surfaces and medical instruments. The disinfectant of theinvention may be useful as a disinfectant for bacteria such as, but notlimited to, Pseudomonadaceae, Azatobacteraceae, Rhizabiaceae,Mthylococcaceae, Halobacteriaceae, Acetobacteraceae, Legionellaceae,Neisseriaceae, and other genera.

The invention also pertains to a method for cleaning and disinfectingcontact lenses. The method includes contacting the contact lenses with asolution of at least one compound of the invention in an acceptablecarrier. The invention also pertains to the solution comprising thecompound, packaged with directions for using the solution to cleancontact lenses.

The invention also includes a method of treating medical indwellingdevices. The method includes contacting at least one compound of theinvention with a medical indwelling device, such as to prevent orsubstantially inhibit the formation of a biofilm. Examples of medicalindwelling devices include catheters, orthopedic devices and implants.

A dentifrice or mouthwash containing the compounds of the invention maybe formulated by adding the compounds of the invention to dentifrice andmouthwash formulations, e.g., as set forth in Remington's PharmaceuticalSciences, 18th Ed., Mack Publishing Co., 1990, Chapter 109 (incorporatedherein by reference in its entirety). The dentifrice may be formulatedas a gel, paste, powder or slurry. The dentifrice may include binders,abrasives, flavoring agents, foaming agents and humectants. Mouthwashformulations are known in the art, and the compounds of the inventionmay be advantageously added to them.

In one embodiment, the invention pertains to each of the transcriptionfactor modulating compounds described herein in Table 4, Table 5, Table6, Table 7, Table 8, Table 9, Table 10, Table 11, and in Formulae(I)-(XVII).

The contents of all references, patent applications and patents, citedthroughout this application are hereby expressly incorporated byreference. Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXEMPLIFICATION OF THE INVENTION Example 1 Synthesis of Test Compounds

The transcriptional modulating compounds described in this applicationcan be synthesized by art recognized techniques or using the methodsdescribed herein.

6-(2-Amino-phenyl)-3-thioxo-3,4-dihydro-2H-[1,2,4]triazin-5-one

This was prepared by a modified literature procedure (Doleschall, G.;Lempert, K. Tetrahedron 1973, 29, 639-649). Isatin (10 g, 67.96 mmol)was dissolved in ca. 10% aqueous KOH (9.9 g in 100 mL of water) and thentreated with thiosemicarbazide (6.28 g; 68.90 mmol). After 1 hour ofheating at 115° C. (bath temperature), the reaction mixture was pouredover ice and treated with glacial acetic acid drop-wise, till the pH wasca. 5. The yellow fluffy precipitate was filtered, washed copiously withwater (8×50 mL) and dried first in air and then under high vacuum toafford 12.9 g of yellow solid.

6-(2-Amino-phenyl)-3-butylsulfanyl-2H-[1,2,4]triazin-5-one

This was prepared by a modified literature procedure (Doleschall, G.;Lempert, K. Tetrahedron 1973, 29, 639-649). The product from theprevious experiment (8.0 g, 36.3 mmol) was dissolved in ca. 10% aq. KOH(10.3 g in 100 mL of water) and treated with ^(n)BuI (7 mL). Ethanol (70mL) was added to it and the reaction mixture was allowed to stirovernight. The reaction mixture was diluted with ether (100 mL) andwater (70 mL). The ether layer was separated and the aqueous layerwashed further with ether (3×100 mL) and then poured over ice. Uponcareful, drop-wise addition of glacial acetic acid with vigorousstirring at 0-4° C., yellow precipitate was obtained which was filtered,washed with water (4×20 mL) and then with ether (2×10 mL) and dried.Yield: 5.12 g.

Other alkyl or substituted alkyl halides were used instead ofn-butyliodide following the similar method.3-Methylsulfanyl-6-(G)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene

This was prepared by a modified literature procedure (Doleschall, G.;Lempert, K. Tetrahedron 1973, 29, 639-649). To a suspension of compound2 (or analogs of 2) (ca. 0.384 mmol, 1 equiv) in dry ethanol (3-4 mL),25 μL of glacial acetic acid was added, followed by ca. 1.1 equiv of thecorresponding aldehyde (G-CHO, where, G=substituted or unsubstitutedaliphatic, aromatic, or heterocyclic groups). The reaction mixture wasrefluxed for ca. 5-7 min resulting in a dark red-dark-reddish orangesolution. Upon cooling to room temperature orange-orange-yellow solidcrashed out of solution, which was filtered, washed with cold (ca. −30°C.) methanol (2×1 mL), and/or ether and dried. In some cases, the crudeproducts were recrystallized from DMF/ether or methanol/ether; in mostof the cases, the crude products, prepared as above, were >95% pure.Various ketones (GCOG′) were reacted with 2 (or analogs of 2) in asimilar way to afford compounds of structural type 4. All the finalcompounds were characterized by means of ¹H NMR, LC-MS, HPLC (C₁₈columns, acetonitrile/water with 0.01% triethylamine as mobile phase),and CHN analyses.

General Synthesis of Orthoesters, GC(OR)₃; R=Me

The syntheses of the desired orthoesters were accomplished by a modifiedliterature procedure in multiple steps (McClelland, R. A. et al. J. Org.Chem. 1981, 46, 1011-1012). Several novel orthoesters were prepared bythis method. To a solution of an acid chloride in dichloromethane,N-methylaniline was added slowly, followed by triethylamine andcatalytic amount of 4-dimethylaminopyridine. After stirring it for ca.12 h, the reaction mixture was diluted with ether, the precipitate wasfiltered, washed with ether and dried. The amide, thus prepared, wasthen stirred overnight with methyl triflate in dichloromethane, dilutedwith ether, and the precipitate was filtered, washed, and dried toobtain an imidatonium triflate salt. This salt was dissolved indichloromethane, cooled to 0° C., and added slowly, with stirring, to acold (0° C.) solution of sodium methoxide in dry methanol over a periodof ca. 30-60 min. The solvent was evaporated to dryness and the residuewas extracted in n-hexane. Upon evaporation of hexane, the white solidwas obtained, which was dissolved in dry methanol and treated withglacial acetic acid. After 10 minutes of stirring, the excess acid wasneutralized with potassium carbonate (solid), and the solvent removedunder vacuum. The residue was extracted in ether, washed with water, anddried over potassium carbonate. The crude material was obtained byevaporation of ether, and further purified either by flashchromatography or fractional distillation.

3-Methylsulfanyl-6-(G)-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c]cycloheptene

Compound of the type 2 (0.384 mmol, 1 equiv) was suspended in ethanol(2-3 mL), treated with glacial acetic acid (100 μL), followed by anorthoester (2 equiv) of the general formula G-C(OR)₃, whereG=substituted or unsubstituted aliphatic, aromatic, or heterocyclicgroup, R=H, substituted or unsubstituted aliphatic, aromatic, orheterocyclic group. The reaction mixture was refluxed for 70-180minutes, cooled to room temperature. In some cases, the product crashedout of solution, in others, the crude reaction mixture was evaporated todryness, re-dissolved in a minimum amount of methanol and diluted withether. The solid was washed with ether (cold, 0-4° C.; 1×1 mL) and driedunder vacuum.

4-Iodo-3-nitrothioanisole

A flask was charged with 10 g of 4-iodothioanisole and 5.5 mL ofdimethylsulfate and warmed to ca. 90° C. for 10 min. The resultingsolution was dissolved in conc. sulfuric acid (30 mL) and cooled to ca.0-4° C., whereupon it was treated, with extreme caution, slowly withconc. HNO₃ (ca. 2 mL) while maintaining the reaction temperature below4° C. After stirring it for ca. 10 min, the reaction mixture was stirredat ca. 90° C. for ca. 3 d. The reaction was monitored with HPLC, TLC,and LC-MS, and if needed, smaller portions of nitric acid were added tothe reaction mixture to force it to completion. Use of fuming sulfuricacid is also helpful. After the complete consumption of the aromaticstarting material, the reaction mixture was cooled, poured over crushedice, treated with 30% aq. perchloric acid at 4° C. The light coloredprecipitate was filtered, washed thoroughly with water, and dried undervacuum. The perchlorate salt was stirred with saturated aq. NaClsolution at 95° C. for 3-6 h. Upon cooling to room temperature, theprecipitate was filtered, washed thoroughly with water to get rid of anyinorganic salts, and dried under vacuum to obtain4-iodo-3-nitrothioanisole in >80% yield.

4-Iodo-3-aminothioanisole

Ca. 7 g of 4-iodo-3-nitrothioanisole was dissolved in absolute ethanoland treated slowly with a solution of SnCl₂.2H₂O in 12% aq. HCl. Thereaction mixture was stirred at 50° C. for 25-30 min., when the HPLCmonitoring indicated that the reaction was complete. The reactionmixture was poured over crushed ice, and treated with aq. NaOH solutionto pH 8. The precipitate was filtered, washed with water and dried inair. The crude material was crystallized by overnight cooling (4° C.) ofits ethanol (minimum amount) solution treated with 10% aq. HCl. Thecrystalline material was further dried under high vacuum to afford thedesired amine as its hydrochloride salt in >70% yield.

4-Methylsulfanyl-2′-nitro-biphenyl-2-ylamine

A methanol/dioxane (20 mL/5 mL of methanol/dioxane) solution/suspensionof 4-iodo-3-nitrothioanisole (1 mmol), Pd(OAc)₂ (0.01 mmol) was purgedwith argon for 5 min. To this solution Et₃N (3 mmol), and 5 mL of waterwere added and purged with argon for another 5 min. To the abovesolution, 2-aminophenyl boronic acid (2 mmol, solution in 5 mL of DMF,purged with argon), was added and the reaction mixture was heated at 70°C. (oil bath temperature) for 2 h. The reaction was monitored by HPLCand LC-MS to follow the product formation. The reaction mixture was thencooled down to room temperature and filtered through diatomaceous earth.The filtrate was concentrated and purified using preparative HPLC.

4-Methylsulfanyl-2′-amino-biphenyl-2-ylamine

A flask was charged with ca. 1 mmol of4-Methylsulfanyl-2′-nitro-biphenyl-2-ylamine, 15 mL of ethanol, and 0.1mmol of PtO₂, and stirred under hydrogen atmosphere at 40 psi for 10minutes. The reaction mixture was filtered through diatomaceous earth,washed with ethanol, and the combined organic layer was evaporated todryness. The crude material was purified by preparative HPLC. The samematerial can also be prepared by the previous method, (Suzuki couplingconditions) starting from 4-iodo-3-aminothioanisole, and purified bypreparative HPLC.

6-(G)-3-methylsulfanyl-5H-dibenzo[d,f][1,3]diazepine

To a solution of the 2,2′-biphenyldiamine (0.093 g; 0.51 mmol) inethanol (2 mL), were added glacial acetic acid (50 μL) and 2 equiv of anorthoester of the general formula GC(OR)₃. In case of TFA salt of thediamine, there was no need of adding acetic acid to the reactionmixture. The reaction mixture was refluxed for 3 h, cooled to roomtemperature, and evaporated to dryness. The residue was suspended inmethanol saturated with dry HCl, stirred for a few minutes, filtered,washed with methanol, and finally with ether. The hydrochloride salt ofthe diazepine was dried under vacuum to afford a light yellow solid. Inorder to obtain a free base of the diazepine, the above hydrochloridesalt was suspended in methanol, and treated with 10% aq. NaOH solution.After stirring at room temperature for ca. 10 min, the precipitate wasfiltered, washed with water, and dried under vacuum.

3-(6-Nitro-2-phenyl-benzoimidazol-1-yl)-propionitrile and3-(5-Nitro-2-phenyl-benzoimidazol-1-yl)-propionitrile

A mixture of 5-nitro-2-phenylbenzimidazole (1 g, 4.2 mmol),acrylonitrile (50 mL) and N,N-dimethylpiridine (25 mg) was heated at 70°C. for 4 hr. The excess of acrylonitrile was evaporated and oily residuewas subjected to the flash chromatography on silica gel usinghexane-ethyl acetate (75:25 v/v) as an eluent. Structure of theregioisomers was determined using ¹H NOESY studies. 0.25 g (20%) of the6-nitro isomer and 0.23 g (18.9%) of the 5-nitro isomer were obtained.

3-(6-Nitro-2-phenyl-benzoimidazol-1-yl)-propionic acid and3-(5-Nitro-2-phenyl-benzoimidazol-1-yl)-propionic acid

To the 6-nitro nitrile (0.15 g, 0.51 mmol) concentrated HCl (5 mL) wasadded and resulting mixture was heated at 50° C. for 0.5 hours. The acidwas evaporated in vacuo and product was purified by HPLC. Yield 34 mg(21%). An identical procedure was used starting from the 5-nitro nitrileyielding the product (22 mg, 13.8%).

Example 2

In this example, the expression of a selective marker (e.g., ccdB) isput under the direct control of a promoter activated by MarA (e.g.,inaA, galT, or micF). In the absence of MarA, the expression of theselective marker is silent and cells survive. Synthesis of MarA from aninducible plasmid in a bacterial or yeast cell leads to the activationof the inaA promoter, expression of ccdB, and subsequently results incell death. Compounds that inhibit MarA are those that permit cellsurvival under conditions of MarA expression. The results of this assayare given in Table 4. In Table 4, * indicates good inhibition of MarAand ** indicates very good inhibition of MarA.

Example 3

In this example, the expression of luciferase is put under the directcontrol of a promoter activated by MarA (e.g., inaA, galT, or micF) in acell constitutively expressing MarA. In the absence of MarA, cellsluminesce. Upon modulating of MarA activity, the expression of thereporter is altered. TABLE 4 ID Structure Name Affinity A

6-(5-Iodo-furan-2-yl)-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza- dibenzo [a,c]cycloheptene ** B

6-(4-Ethoxy-phenyl)-3-methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza- dibenzo [a,c]cycloheptene * C

3-Methylsulfanyl-6-(5-nitro-furan-2-yl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza- dibenzo [a,c]cycloheptene * D

3-Methylsulfanyl-6-[5-(4-nitro-phenyl)-furan-2-yl]-6,7-dihydro-5-oxa-1,2,4,7- tetraaza-dibenzo[a,c]cycloheptene * E

4-(3-Ethylsulfanyl-6,7-dihydro-5-oxa- 1,2,4,7-tetraaza-dibenzo[a,c]cyclohepten-6-yI)-benzene-1,2-diol * F

6-(4-Benzyloxy-phenyl)-3- propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo [a,c]cycloheptene ** G

6-Benzo[1,3]dioxol-5-yl-3- methylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo [a,c]cycloheptene ??? H

3-Butylsulfanyl-6-(2,4-dimethoxy- phenyl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo[a,c] cycloheptene * I

6-(4-Allyloxy-phenyl)-3-butylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza- dibenzo[a,c]cycloheptene ** J

3-Butylsulfanyl-6-(4-ethoxy-phenyl)-6,7-dihydro-5-oxa-1,2,4,7-tetraaza-dibenzo [a,c] cycloheptene * K

6-(4-Methoxy-phenyl)-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7-tetraaza- dibenzo [a,c]cycloheptene * L

6-[5-(3-Nitro-phenyl)-furan-2-yI]-3-propylsulfanyl-6,7-dihydro-5-oxa-1,2,4,7- tetraaza-dibenzo[a,c]cycloheptene ** M

2-(3-Phenyl-1H-pyrazol-4-ylmethylene)- benzo[4,5]imidazo[2,1-b]thiazol-3-one ** N

2-[5-(3-Carboxy-phenyl)-furan-2- ylmethylene]-5-(2-methoxy-naphthalen-1-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester ** O

5-(4-Dimethylamino-phenyl)-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a] pyrimidine-6-carboxylic acid ethyl ester *** P

5-Benzo[1,3]dioxol-5-yl-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a] pyriniidine-6-carboxylic acid ethyl ester * Q

5-(3,4-Dimethoxy-phenyl)-7-methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-yl methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester *** R

7-Methyl-2-[5-(2-methyl-4-nitro-phenyl)-furan-2-ylmethylene]-5-(4-methyl sulfanyl-phenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a] pyrimidine-6-carboxylic acid ethyl ester *** S

2-[5-(4-Carboxy-phenyl)-furan-2- ylmethylene]-5-(2-methoxy-naphthalen-1-yl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester *** T

5-Benzo[1,3]dioxol-5-yl-2-[5-(4- ethoxycarbonyl-phenyl)-furan-2-ylmethylene]-7-methyl-3-oxo-2,3- dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester *** U

7-Methyl-3-oxo-5-phenyl-2-[5-(3- trifluoromethyl-phenyl)-furan-2-ylmethylene]-2,3-dihydro-5H- thiazolo[3,2-a]pyrimidine-6-carboxylic acidethyl ester ** V

7-Methyl-2-[5-(2-methyl-4-nitro-phenyl)- furan-2-ylmethylene]-3-oxo-5-phenyl- 2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester *** W

2-[5-(3-Carboxy-phenyl)-furan-2- ylmethylene]-5-(4-dimethylamino-phenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester ** X

5-(4-Dimethylamino-phenyl)-7-methyl-2-[5-(4-methyl-3-nitro-phenyl)-furan-2-yl methylene]-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester ** Y

2-[5-(3-Carboxy-phenyl)-furan-2- ylmethylene]-7-methyl-5-(4-methylsulfanyl-phenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6- carboxylic acid ethyl ester ***Z

[1,2]Naphthoquinone 1-[O-(6-oxo-6H- anthra[1,9-cd]isoxazol-5-yl)-oxime] * AA

3-Acetyl-2,5,7-triphenyl-1H-1,3a,4,8-tetraaza-7a-azonia-cyclopenta[a]indene *** AB

1-Amino-3-benzo[1,3]dioxol-5-yl- benzo[4,5]imidazo[1,2-a] pyridine-2,4-dicarbonitrile ** AC

2-[2-(5-Furan-2-yl-4-phenyl-4H- [1,2,4]triazol-3-ylsulfanyl)-acetylamino]- benzoic acid methyl ester * AD

6,7-Dimethyl-2-(3-phenyl-1H-pyrazol-4-ylmethylene)-benzo[4,5]imidazo[2,1-b]thiazol-3-one * AE

2-(5-Benzo[1,2,5]oxadiazol-5-yl-4- methyl-4H-[1,2,4]triazol-3-ylsulfanyl)- N-(3-methylsulfanyl-phenyl)-acetamide * AF

4-(1,3-Dioxo-indan-2-ylidene)-2-phenyl-6-pyridin-2-yl-tetrahydro-pyrrolo[3,4-c]pyrrole-1,3-dione ** AG

6-Nitro-2-phenyl-1-(3-trifluoromethyl- benzyloxy)-1H-benzoimidazole **AH

(6-Nitro-2-phenyl-benzoimidazol-1- yloxy)-acetic acid ** AI

1-Benzyloxy-6-nitro-2-phenyl-1H- benzoimidazole ** AJ

1-(4-Methyl-benzyloxy)-6-nitro-2- phenyl-1H-benzoimidazole * AK

6,8-Dimethyl-2-(4-nitro-phenyl)-5- phenyl-5H,6H-1-oxa-3,5,6,8-tetraaza-cyclopenta[a]naphthalene-4,7,9-trione ** AL

6,8-Dimethyl-5-phenyl-2-p-tolyl-5H,6H- 1-oxa-3,5,6,8-tetraaza-cyclopenta[a]naphthalene-4,7,9-trione * AM

2-[3-(4-Fluoro-phenyl)-1-phenyl-1H- pyrazol-4-yl methylene]-benzo[4,5]imidazo[2,1-b]thiazol-3-one ** AN

Cobalt 5,10,15,20-Tetra-pyridin-4-yl- porphyrine *** AO

2-[3-(4-Fluoro-phenyl)-1-phenyl-1H- pyrazol-4-ylmethylene]-5-methyl-6-vinyl-imidazo[2,1-b]thiazol-3-one ** AP

Cobalt 5,10,15,20-Tetra-pyridin-3-yl- porphyrine *** AQ

Zinc 5,10,15,20-Tetra-pyridin-4-yl- porphyrine *** AR

2-(4-isopropylphenyl)-4H-chromen-4-one *** AS

2-(3,4-Dihydroxy-phenyl)-3,5,7- trihydroxy-chromen-4-one (luteolin) ***AT

N-isopropyl-2-[(4-methyl-5-quinolin-6-yl-4H-1,2,4-triazol-3-yl)thio]acetamide *** AU

4-hydroxy-6-methyl-5,6-dihydro-2H- pyrano[3,2-c]quinaline-2,5-dione ***AV

5,7-Dihydroxy-2-(4-hydroxy-phenyl)- chromen-4-one *** AW

2-[4-(dimethylamino)phenyl]-4H- chromen-4-one ** AX

1-(benzyloxy)-2-phenyl-1H-imidazo[4,5- b]pyridine ** AY

2-(benzylthio)-4-phenyl-5-(1-phenyl-1H-1,2,3,4-tetraazol-5-yl)pyrimidine ** AZ

6-fluoro-2-phenyl-4H-chromen-4-one ** BA

7-methoxy-2-phenyl-4H-chromen-4-one * BB

4-(1,3-dioxo-1,3-dihydro-2H-inden-2- yliden)-2-phenyl-6-(2-pyridinyl)tetrahydro pyrrolo [3,4-c]pyrrole- 1,3(2H,3aH)-dione * BC

2-(2-Hydroxy-3-oxo-5-p-tolyl-2,3- dihydro-furan-2-yl)-malonamic acidethyl ester * BD

2-[(6-nitro-2-phenyl-1H-1,3- benzimidazol-1-yl)oxy]acetic acid * BE

2-(4-fluorophenyl)-4H-chromen-4-one * BF

1-methoxy-2-(4-methyl phenyl)-1H- imidazo [4,5-b] pyridine * BG

2-(4-hydroxyphenyl)-4H-chromen-4-one *

Example 4

In this example, the expression of a selective marker (e.g., ccdB) isput under the direct control of a promoter repressed by MarA (e.g.,fecA, purA, guaB). Under conditions of constitutive MarA synthesis(e.g., using a constitutive mar (marc) mutant), the expression of ccdBis silent. Following inactivation of MarA, the synthesis of ccdB resultsin cell death.

Example 5

In this example, the expression of a selective marker (e.g., URA3) isput under the direct control of a promoter repressed by MarA (e.g.,fecA, purA, guaB). Under conditions of constitutive MarA synthesis(e.g., using a constitutive mar (marc) mutant), the expression of URA3is silent. Following inactivation of MarA, and in the presence of 5-FOAthe synthesis of URA3 results in cell death.

Example 6

In this example, a purine or guanine heterotroph is constructed byinactivation of either of the chromosomal guaB or purA genes in E. coli.The guaB or purA gene is then placed into a suitable vector under thecontrol of its natural promoter and transformed into the heterotrophichost.

Example 7 E. coli Bioflim Assay

The biofilm assay screens test compounds for their ability to inhibitbacteria from forming a biofilm.

Materials:

The M9 media (“M9”) contained M9, casamino acids, and glucose. The testcompound was dissolved in 10 mg/mL DMSO stock solution.

Method:

Preparation of Inoculum Inoculum was started the day of the experimentby adding a colony or glycerol stock stab to 2 mL M9. The tube wasplaced in the 37° C. shaker incubator for approximately 4-6 hours. Thisinoculum was referred to as the “Starter inoculum.” The inoculum wasthen removed from the shaker incubator and diluted to 1×10⁶ cells/mL inM9.

Preparation of Controls

Typically, there were eight of each control, including a positive andnegative control. For both the positive and negative controls, 2.5 μL ofDMSO was added to 200 μL of M9. 25 μL of the diluted DMSO was added to50 μL of M9 in the assay plate.

Preparation of Test Compounds

The test compounds were screened at 20 μg/mL. 2.5 μL of the testcompound were taken from a plate containing 10 mg/mL stock and added to200 μL of M9 and mixed. 25 μL of the diluted test compound was added to50 μL of M9 in the assay plate. The resulting concentration of the testcompound was 40 μg/mL

Preparation of Plate

75 μL of the inoculum at 1×10⁶ cells/mL was added to each wellcontaining compound and the positive controls. 75 μL M9 was added to thenegative controls. The final concentration of the test compound was 20μg/mL and the final concentration of the inoculum was 2×10⁵ cells/mL.The plates were then placed in a microplate reader (Wallac Victor²V) andread OD₅₃₅ (“Initial growth reading”). The plates were then placed in anincubator overnight at 35° C. In the morning, the plates were read in amicroplate reader at OD₅₃₅ (“Final growth reading.”)

Addition of Crystal Violet

The inoculum was then removed from the wells and the plates were washedseveral times with tap water. 150 μL of Crystal Violet (0.02% CrystalViolet dissolved in water) was then added to each well.

Addition of Ethanol

The crystal violet was then removed and the plates were washed severaltimes with tap water. 150 μL of ethanol were then added to each well,after mixing. The plates were then placed in a microplate reader andread the OD₅₃₅. This was referred to as the “Crystal Violet” reading.

Data Analysis

To determine whether a test compound inhibited growth, the Initialgrowth reading was subtracted from the Final growth reading (“SubtractedGrowth”). The same was done for the positive controls and averaged. The% inhibition of growth was determined by the following formula:100−(100*Subtracted growth of sample/Average growth of Pos Controls)

To determine whether a test compound inhibited Biofilm formation, the %Inhibition of Biofilm Formation was determined using the followingformula:100−(100*Crystal Violet read of sample/Average crystal violet read ofPos Controls)

The results from the Crystal Violet assay are summarized in Table 5. InTable 5, ND indicates that a given compound did not inhibit biofilmformation in the CV assay. * indicates that the test compound inhibitedsome biofilm formation and ** indicates that the compound inhibited theformation of a biofilm well.

Example 8 LANCE Screening Assay for Inhibitors of MarA, SoxS, or RobDNA-Binding

This example describes a method for the identification of test compoundsthat inhibit the interactions of purified transcription factor such asMarA, SoxS and/or Rob with a target DNA sequence in an in vitro system.Such molecules will hopefully be able to inhibit this interaction invivo, leading to inhibition of transcriptional regulation by thesefactors and ultimately in inhibition of the drug resistance and/orvirulence phenotypes associated with MarA, SoxS and Rob.

Materials

The 6His-tagged MarA, SoxS and Rob purified according to respectiveprotocol. The N-term-biotinylated double-stranded DNA has a sequence ofCCG ATT TAG CAA AAC GTG GCA TCG GTC (SEQ ID NO. 5). The antibody usedwas the LANCE Eu-labeled anti-6×His Antibody (Eu-αHis) (Perkin Elmer cat# AD0110) which had at least 6 Europium molecules per antibody.Streptavidin conjugated to SureLight™-Allophycocyanin (SA-APC) wasobtained from Perkin Elmer (cat # CR130-100). The Assay buffer contained20 mM Hepes pH 7.6, 1 mM EDTA, 10 mM (NH₄)₂SO₄, and 30 mM KCl, and 0.2%Tween-20.

Method

The plates or vials of the compounds to be tested were thawed. Thesestocks were at a concentration of 10 mg/ml in DMSO. The solutions wereallowed to thaw completely, and the plates were briefly shaken on theTitermix to redissolve any precipitated compound. Thawed aliquots ofMarA, SoxS and Rob protein from the stock stored at −80° C. and 1M stockof dithiothreitol stored at −20° C. were then placed on ice.

Dilutions at 1:100 of the compounds were made into a fresh 96-wellpolystyrene plate. The dilutions were prepared with 100% DMSO to give afinal concentration of 100 μg/ml solutions. The dilutions were vortexedon a Titermix.

Fresh DTT was added to 25-50 mL of assay buffer to produce a 1 mM finalconcentration. Next, 90 μl of assay buffer was added to each of the 10μl protein aliquots, and the solution was mixed by pipetting. Theseproteins were diluted to give the required amount of each of the dilutedproteins, resulting in 20 μl of diluted protein per well. In preparingthe solutions, 20% excess was made to allow enough for control wells.Typically, depending on the protein preps and the initial binding curvesthat were performed, 1000-2000 fmoles of each protein was required perwell. The diluted protein solutions were the placed on ice.

Three tests plates per plate of compound (for MarA, SoxS and Rob) wereprepared. Using a multichannel pipet, 5 μl of the compound was added toeach well. 5 μl of DMSO was added to the blank and control wells, and 5μl of the control inhibitor was added to the respective wells.

Using the multichannel pipet, 20 μl of protein was added to all wellsexcept those designated “blank”. To these blank wells, 20 μl of assaybuffer was added. The plates were covered with a plate sealer andincubated at room temperature, shaking on the Titermix, for 30 minutes.

Next, the DNA solution was prepared, with enough for at least 20% morewells than were tested. 15 μl (0.4 fmoles) was added per well. Then theDNA was diluted in assay buffer, and vortexed briefly to mix. The platesealer was removed, and 15 μl of DNA solution was added to all of thewells. the plates were then resealed, and returned to the Titermix for afurther 30 minutes.

After 25 minutes, the antibody solution was prepared. 0.4 fmoles ofSA-APC and 0.125 fmoles of Eu-αHis were added per well in a total volumeof 10 μl. Amounts were prepared sufficient for at least 20% excess. Theplate sealer was the removed and 10 μl of antibody solution was added toevery well. The plates were subsequently resealed, placed on theTitermix, and covered with aluminum foil. The plates were mixed for 1hour. The plates were then read on the Wallac Victor V, using the LANCE615/665 protocol.

Data Processing

For each plate, the mean control (i.e. signal from protein and DNAwithout inhibition), mean blank (background signal without protein) andmean inhibitor (P001407) LANCE₆₆₅ counts were determined. The percentageinhibition by each molecule (each test well) was then determinedaccording to the following equation:% Inhibition=100−(((test−mean blank)/(mean control−mean blank)*100)

Compounds that gave 40% or greater inhibition were identified as hitsand screened again for IC50.

IC₅₀ Screening

The protocol used was identical to that outlined above, except that only10 compounds were assayed per plate. The testing concentrations startedat 10 μg/ml and were diluted two-fold from 10 to 0.078 μg/ml.

IC₅₀ Data Processing

Percent inhibition was calculated as shown above. Percent inhibition wasthen plotted vs. log (conc. Inhibitor) using Graph pad Prism software.The IC₅₀ concentration was determined as the concentration that gives50% inhibition.

The data from this example is also summarized in Table 5. *** indicatesthat a particular test compound inhibited the particular bacteria verywell, ** indicates that the particular test compound inhibited theparticular bacteria well, and * indicates that the particular testcompound inhibited the particular bacteria to some extent. TABLE 5 IDSTRUCTURE BH

BI

BJ

BK

BL

BN

BO

BP

BQ

BR

BS

BT

BU

BV

BW

BX

BY

BZ

CA

CB

CC

CD

CE

CF

CG

CH

CI

CJ

CK

CL

CM

CN

CO

CP

CQ

CR

CS

CT

CU

CV

CW

CX

CY

CZ

DA

DB

DC

DD

DE

DF

DG

DH

DI

DJ

DK

DL

DM

DN

DO

DP

DQ

DR

DS

DT

DU

DV

DW

DX

DY

DZ

EA

EB

EC

ED

EF

EG

EH

EI

EJ

EK

EL

EM

EN

EO

EQ

ER

ES

ET

EU

EV

EW

EX

EY

EZ

FA

FB

FC

FD

FE

FG

FH

FI

FJ

FK

FL

FM

FN

FO

FP

FQ

FR

FS

FT

FU

FV

FW

FX

FY

FZ

GA

GB

GC

GD

GE

GF

GG

GH

GI

GJ

GK

GL

GM

GN

GO

GP

GQ

GR

GS

GT

GU

GV

GW

GX

GY

GZ

HA

HB

HC

HD

HE

HF

HG

HH

HI

HJ

HK

HL

HM

HN

HO

HP

HQ

HR

HS

HT

HU

HV

HW

HX

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Mar A Mar A Sec3 Sec3 Rab Rab ID IGEN Lance IGEN Lance IGEN Lance OO CVLen BH * ** ** * * ** ** ** ** BI ** ** ** NT * NT ** ** ND BJ *** ***** *** *** * ** ND ** BK * * ** * *** ** ** ND ND BL * * ** ** * * ** **ND BN NT * *** * * * ** ** ND BO * * ** ** * * ** ** ND BP * * ** NT *NT ** * ** BQ ** * * * ** *** ** ND ** BR *** *** *** *** *** *** ND NDBS NT * NT * NT * ** ** ** BT NT * ** ** ** * ** * * BU * * ** NT * NT** ND ND BV * * ** NT * NT ** ** ** BW * NT ** *** ** ** ** ND NDBX * * * NT ** NT ** ND ND BY ** * * * *** *** ** ND ** BZ *** *** ** *** ** ** ND ND CA * *** ** ** * NT ** ND ** CB * * ** * ** * ** ND **CC * * ** * ** ** ** * ND CD * NT ** *** * *** ** ND ** CE NT ** NT **NT * ** * ND CF * * ** NT ** NT ** ND ** CG * * ** NT ** NT ** ND **CH * * * NT * NT ** ND ND CI * * ** NT ** NT ** ND ** CJ ** ** * * * *** ND ND CK ** ** * *** *** ** ** ND ND CL * ** ** * ** * ** ND NDCM * * ** * * * ** ** ** CN *** *** *** *** ** * ** ** ND CO * * ** * *** ** ** * CP ** * *** * *** * ** ** * CQ NT * ** * * * ** ** * CR NT *NT ** NT *** ** ** ND CS NT *** NT *** NT * ** ** ND CT ** NT *** *** ***** ** ND * CU NT NT ** ** * * ** ND ND CV NT * * NT ** NT ** ND ND CW*** * ** * *** *** ** ND ** CX * * ** *** ** *** ** ND ** CY *** * * *** * ** ND ND CZ ** * * * * ** ** ND ND DA *** * * * * * ** ND ND DB ***** * * * * ** ND ND DC ** * ** * ** * ** ND * DD ** * * * * * ** ND * DENT * NT * NT ** ** ** ND DF ** * * * ** * ** ND ** DG * * ** * * * ** **** DH * * ** * * ** ** ** ND DI ** * ** * * * ** ** ** DJ * ** * NT **NT ** ND ND DK * * ** NT ** NT ** ND ND DL *** *** * * ** *** ** ND **DM *** *** *** * *** * ** ND ND DN * * ** * ** * ** ND ND DO * * ** *** * ** ND * DP *** *** * * * * ** ND ND DQ ** * * * * * ** ND ND DR ***** * * * * ** ND ND DS *** * * * ** * ** ND * DT *** * ** * ** * **ND * DU *** *** *** *** *** * ** ND ** DV *** *** *** *** *** ** ** ND** DW *** ** ** * ** ** ** ND ** DX *** *** *** *** *** ** ** ND ** DY** * ** * ** ** ** ND ** DZ ** * NT * ** ** ** ** ** EA * * * NT * NT** * * EB * * ** NT ** NT ** * ** EC * * ** NT * NT ** ** ND ED NT ***NT *** NT * ** ** ** EF NT ** NT *** NT *** ** * ** EG NT * NT ** NT *** ** ** EH NT *** NT *** NT ** ** * ** EI * * * NT ** NT ** ND NDEJ * * * NT * NT ** ND ND EK * *** ** NT ** NT ** ND * EL * * * NT * NT** ND ND EM NT ** ** NT * NT ** ND ND EN * * ** NT ** NT ** ND * EO ** *** * ** * ** ND ** EQ * * ** * ** * ** ND ND ER * * ** * ** * ** ND **ES * * ** * ** *** ** ND ND ET *** ** *** * ** * ** ND ** EU * * * * * *** ND ND EV *** *** *** * * * ** ND ND EW * * * * * ** ** ND ** EX*** * * * *** ** ** ND ND EY ** * * * * * ** ND * EZ ** * * * ** * **ND * FA ** * * * * * ** ND * FB ** * * * * * ** ND * FC NT * NT * NT *** * * FD NT * NT * NT * ** * ** FE NT * NT ** NT * ** ** ND FG NT ** NT*** NT ** ** * ND FH * * ** * ** * ** ND ** FI ** * * * * * ** ND ND FJ** ** *** ** ** * ** ND ** FK *** *** *** * *** *** ** ND ** FL ** *** * ** ** ** ND ** FM *** * * * ** * ** ND ** FN * *** * ** ** NT **ND * FO *** *** *** ** *** * ** * * FP *** *** *** *** ** * ** ** **FQ * * ** ** * ** ** ** * FR * * * NT ** NT ** * ** FS NT * NT ** NT *** ** ND FT * NT ** * * * ** ND ** FU ** ** ** * ** *** ** ND ** FV * *** * ** * ** ND ** FW ** * ** * *** * ** ND ND FX * * ** * ** * ** ND **FY ** * *** * ** * ** ND ** FZ * * ** * ** * ** ND ** GA * * ** * *** **** ND ND GB ** * *** * ** * ** ND * GC ** * ** * ** * ** ND **GD * * * * * * ** ND ND GE * * * * * * ** ND ND GF ** ** *** * * * ** NDND GG * * * * ** * ** ND * GH * * * * * * ** ND ND GI ** ** ** ** ** ***** ND ** GJ ** * * * * ** ** ND ND GK ** * * * * * ** ND * GL** * * * * * ** ND ND GM ** * * * * *** ** ND ND GN ** * * * * * ** NDND GO ** * * * ** * ** ND * GP NT * NT ** NT ** ** ** ** GQ NT ** NT ***NT ** ** * ** GR NT * NT *** NT *** ** * ND GS NT *** NT *** NT *** **** ND GT NT * NT ** NT ** ** * ND GU NT ** NT *** NT * ** ** ND GV** * * * * * ** ND ND GW *** * ** * ** ** ** ND ** GX ** * ** * *** ***** ND ** GY ** * * * ** * ** ND ** GZ ** ** ** * ** ** ** ND ** HA *** **** * *** * ** ND ** HB * * ** * ** * ** ND * HC * *** ** * ** NT ** ND** HD * ** ** * ** *** * ND * HE * * ** * * * ** ** ** HF * ** ** * * *** ** ND HG * * ** * * * ** ** ** HH * * ** * ** * ** ** ** HI * * ** *** * ** ** ** HJ NT * ** ** * * ** ** ** HK NT * NT ** NT ** ** ** * HLNT *** NT *** NT * ** ** ** HM * NT ** ** * * ** ND ** HN * NT ** ** *** ** ND ND HO ** NT ** * * ** ** ND ND HP * NT ** * * * ** ND * HQ NT *NT ** NT * ** ** ** HR NT * NT * NT * ** ** ** HS * ** ** NT ** NT **ND * HT * * * NT ** NT ** ND * HU *** *** *** *** *** * ** ND ** HV ** **** * ** * ** ND ** HW *** * *** *** ** * ** ND ** HX * * *** * ** * **ND ** HY *** * *** * *** * ** ND * HZ ** * ** * ** * ** ND ** IA *** **** * *** * ** ND * IB ** * *** * ** * ** ND ND IC ** * ** * *** * ** NDND ID *** * *** * *** * ** ND * IE *** *** *** *** *** * ** ND ** IF * **** * ** * ** ND ND IG * * ** * ** * ** ND ND IH ** * *** * ** * ** ND *II *** ** *** * ** * ** ND ** IJ *** ** *** * *** * ** ND * IK *** *** * *** * ** ND ND IL *** *** *** * *** * ** ND ** IM ** * ** * ** * **ND ** IN ** * * * ** * ** ND ND IO * * * * * * ** ND ND IP * * * * ** *** ND ND IQ *** * ** * *** * ** ND ND IR ** * ** * ** ** ** ND ** IS** * * *** ** ** ** ND * IT ** * * * ** * ** ND * IU ** *** * * * ** **ND ND IV * * * * * * ** ND ND IW *** * * * * * ** ND ND IX ** ** * * * *** ND * IY ** * * * * * ** ND ** IZ ** * * * * * ** ND * JA ** * * *** * ** ND * JB *** ** ** * *** * ** ND ND JD ** * * * * * ND * JE NT *NT * NT * ** ** * JF NT ** NT ** NT *** ** * * JG NT * NT * NT ** ** **ND JH NT * NT * NT * ** ** ** JI NT * NT * NT ** ** ** ** JJ ** ** ** *** ** ** ND ** JK ** ** * * ** * ** ND ** JL * ** ** * *** * ** ND NDJM * * * * * * ** ND ** JN * *** * * * NT ** ND ** JO ** *** ** * ** NT** ND ** JP * * ** * ** * ** * * JQ * * ** * ** * ** ** ** JR *** ****** *** * *** ** ** ND JZ * ** ** * * * ** ** ** KA * * NT * * * ** **** KB *** * *** * * * ** ** ** KC * * ** * * * ** ** ** KD ** * ** NT *NT ** * ** KE * * * NT ** NT ** ** * KF NT *** NT *** NT ** ** ** **KG * NT ** * * * ** ND * KH * NT ** * ** *** ** ND ** KI * NT *** * * *** ND ND KJ * NT ** ** * *** ** ND ND KK * * * NT * NT ** ND ** KL ** *** * ** * ** ND ND KM ** * ** * ** ** ** ND ND KN * *** ** * ** * ** ND** KO ** * *** *** ** *** ** ND ** KP * * ** * ** ** ** ND ** KQ * *** * *** * ** ND * KR *** *** *** ** *** * ** ND ND KS * * ** * ** * **ND * KT ** * *** * *** * ** ND * KU ** * *** * *** ** ** ND ** KV *** **** * ** * ** ND * KW * * * * * * ** ND ND KX ** * * ** * * ** ND ND KY*** * * ** ** * ** ND ** KZ ** ** * * * ** ** ND ND LA *** * * * * * **ND ND LB ** * * * * * ** ND ND LC * * * * * * ** ND ND LD ** * * * * *** ND * LE *** * * * * * ** ND ND LF ** * * * * ** ** ND ND LG ** * * *** * ** ND * LH *** ** * * * * ** ND * LI *** * ** * *** * ** ND * LJNT * NT ** NT * ** * ** LK NT * NT * NT * ** ** * LL NT * NT * NT * **** * LM NT ** NT *** NT * ** ** ND LN ** ** ** * *** * ** ND ** LO ****** *** *** *** ** ** ND ** LP *** *** *** * ** * ** ND * LQ ** ** * **** *** ** ND * LR ** * * * ** * ** ND ** LS *** ** *** ** ** * ** ND *LT * ** *** ** ** ** ** ND ND LU * *** ** *** ** NT ** ND ND LV * *** * * ** ** ** * LW * * NT * ** * ** ** ** LX *** *** NT * * * ** ** **LY * * ** * ** * ** ** ** LZ * * NT * * * ** ** ** MA * * ** NT * NT** * * MB * * * NT ** NT ** ** ND MC * * ** ** * * ** * * MD NT *** NT*** NT ** ** ** * ME NT *** NT *** NT ** ** ** * MF NT *** NT *** NT ***** ** ND MG NT * NT ** NT ** ** ** * MH NT *** NT *** NT *** ** ** *MI * NT * * * * ** ND ** MJ * NT * * * * ** ND * MK ** NT ** ** * ** **ND ND ML * NT ** ** * *** ** ND ** MM * NT ** *** * *** ** ND ND MN NT *NT ** NT * ** * ND MO NT * NT * NT * ** ** ** MP NT * NT ** NT * ** **** MQ NT * NT *** NT * ** ** ND MR * ** ** NT * NT ** ND ** MS NT * *NT * NT ** ND ND MU ** ** *** * ** * ** ND * MV * * ** * ** * ** ND **MX *** *** *** *** *** ** ** ND ND MY ** *** ** * ** * ** ND ** MZ * *** * *** * ** ND * NA * *** ** * ** * ** ND * NB * ** * * ** ** ** ND *NC * * *** * ** * ** ND * ND *** *** *** * *** * ** ND ND NE ** * ** *** * ** ND ** NG ** * *** * ** * ** ND ** NH ** * * * * * ** ND NDNI * * * * * * ** ND ND NJ * * * * ** * ** ND ND NK * * * * * * ** ND NDNL ** * * * ** *** ** ND * NM * * * ** * * ** ND ** NN ** * * * * ** **ND * NO *** * * ** ** ** ** ND * NP *** * * ** *** * ** ND ND NQ** * * * * * ** ND ND NR ** * * * ** * ** ND ND NS ** * * * * * ** ND NDNT ** * * * * * ** ND ND NU ** * * * * * ** ND ND NV * * * * * * ** NDND NW ** * * * * * ** ND * NX ** ** * * * * ** ND ** NY ** * * * * * **ND * NZ *** * * * *** * ** ND ND OA *** *** ** *** ** * ** ND ND OB*** * ** * ** * ** ND ** OC *** ** ** * ** * ** ND ND OD *** *** ** **** ** ** ND * OE *** * ** * *** * ** ND ND OF NT * NT * NT *** ** * NDOG NT * NT * NT * ** * ** OH NT * NT * NT * ** * ** OI NT * NT ** NT **** ** ND OJ NT * NT ** NT ** ** * ND OK NT *** NT *** NT ** ** ** ND OLNT * NT ** NT *** ** ** * OM NT * NT ** NT *** ** * ND ON ** ** ** * ***** ** ND ** OO * * ** * ** * ** ND * OP * * ** * ** * ** ND ** OQ ***** ** * ** * ** ND * OR * ** * * * * ** ND ** OS * ** ** * * * ** ND *OT * ** ** ** ** ** ** ND * OU ** * ** ** *** ** ** ND ND OV * *** **** * NT ** ND ** OW * * * * * ** ** ** ND OX * * ** * * * ** ** * OY * *** * * * ** ** ND OZ * * ** * ** * ** * ** PA *** *** *** NT * NT **** * PB NT *** NT *** NT ** ** ** ND PC NT ** NT *** NT * ** * ** PD **NT ** *** ** * ** ND ND PD * NT ** *** ** * ** ND ** PE * NT ** ** ** *** ND ND PF ** NT *** *** *** ** ** ND * PG * NT ** * * * ** ND ND PH *NT ** * ** * ** ND ND PI ** NT *** ** * * ** ND ND PJ * NT ** * ** ** **ND ** PK NT * NT ** NT * ** ** ** PL NT * NT ** NT * ** ** ** PM NT * NT** NT * ** * ** PN NT * NT ** NT ** ** ** * PO NT * NT * NT * ** * ND PP*** * * NT * NT ** ND ND PQ ** ** ** NT ** NT ** ND ND PR ** *** * NT *NT ** ND ND PS * * ** NT * NT ** ND ** PT ** * ** * ** * ** ND ND PU*** * *** * ** * ** ND ** PV *** * *** * *** * ** ND ** PW ** * *** **** * ** ND * PX * * ** * ** * ** ND ** PY * * *** * ** * ** ND ** PZ** * ** * ** * ** ND ** QA * * ** * ** ** ** ND ** QB ** *** *** * ***** ** ND * QC *** *** *** * *** * ** ND ND QD ** * ** * ** * ** ND NDQE * * *** * ** * ** ND * QF *** *** *** * *** * ** ND ND QG ** * ** *** * ** ND ** QH *** * *** * *** * ** ND * QI *** *** *** *** *** *** **ND * QJ ** * *** * *** ** ** ND * QK ** * ** * ** * ** ND ** QL ***** * * ** * ** ND ND QM * * * * * * ** ND ND QN * * * * * * ** ND NDQO * * * * ** * ** ND * QP *** * * * *** * ** ND * QQ * * * * ** ** **ND ND QR ** ** * * * *** ** ND ND QS * * ** ** * *** ** ND ND QT** * * * ** *** ** ND ND QU ** ** * ** * ** ** ND * QV ** * * ** ** **** ND * QW *** * * * * ** ** ND ND QX ** ** * * * ** ** ND ND QY** * * * * ** ** ND ND QZ ** ** * * * * ** ND ND RA ** ** * * * ** ** ND** RB ** * * * * * ** ND * RC ** * * ** * * ** ND ND RD NT ** NT *** NT*** ** * ND RE NT * NT ** NT *** ** * ND RF NT ** NT *** NT ** ** * **RG NT * NT * NT ** ** * ** RH NT * NT * NT ** ** * ** RI NT * NT * NT *** * ND RJ NT ** NT ** NT * ** * ** RK NT * NT *** NT ** ** * ** RL NT** NT *** NT ** ** ** ND RM NT * NT * NT * ** * ND RN NT * NT * NT **** * * RO NT * NT * NT ** ** * * RP NT ** NT ** NT *** ** * ** RQ NT ***NT *** NT * ** * ** RS *** *** *** * *** *** ** ND ** RT *** *** *** ***** *** ** ND * RU *** *** * ** * ** ** ND * RV *** ** *** * ** * ** ND** RW *** ** ** ** *** * ** ND ** RX *** ** *** *** *** * ** ND ND RY **** ** * ** * ** ND ** RZ * * ** * ** * ** ND ** SA ** * ** * ** * **ND * SB * ** ** * ** * ** ND ** SC * *** ** * ** * ** ND ** SD * * **** * * ** ND * SE *** ** ** * ** * ** ND ND SF *** * ** * ** ** ** NDND SG * ** ** * ** NT ** ND * SH ** ** *** ** ** NT ** ND ND SI * ** ***** * NT ** ND ND SJ *** * *** *** * * ** ** ND SK * * ** * * * ** ** NDSL * * ** * ** * ** ** ND SM * * ** NT ** NT ** * ** SN * * ** NT ** NT** * * SO * * * NT ** NT ** ** * SP * NT ** ** * ** ** ND ** SQ * NT **** * *** ** ND ** SR * NT ** * ** * ** ND * SS * NT ** * ** * ** ND NDST * NT * ** * * ** ND ** SU * NT * * * * ** ND ** SV NT ** NT ** NT *** ** ** SW NT * NT * NT ** ** ** * SX * * * NT ** NT ** ND * SY ** ***** * *** * ** ND ND SZ ** * ** * ** * ** ND ND TA ** * *** * ** * **ND * TB * * ** * ** * ** ND ** TC *** * *** * *** * ** ND ND TD *** **** * *** * ** ND * TE * * *** * ** * ** ND ** TF * * ** * ** * ** ND **TG ** *** ** * ** ** ** ND ** TH * * ** * ** * ** ND ** TI * * *** * **** ** ND * TJ * * * * ** * ** ND ** TK ** * *** ** ** *** ** ND NDTL * * ** * ** * ** ND ND TM *** *** *** *** *** * ** ND * TN * * * *** * ** ND ND TO * * NT * * *** ** ND * TP *** * * * *** ** ** ND ND TQ*** * * ** *** *** ** ND * TR *** * * ** *** ** ** ND ND TS *** * * ***** * ** ND ND TT *** * *** *** *** *** ** ND ND TU ** ** * * * * **ND * TV *** * * * ** * ** ND * TW *** * * * *** * ** ND * TX *** ** * **** * ** ND * TY *** * * * ** * ** ND ND TZ *** * * * *** * ** ND ND UA*** * * * *** * ** ND ND UB NT ** NT * NT * ** * * UC NT ** NT *** NT*** ** ** ** UD NT ** NT *** NT *** ** * ** UE * * ** * ** ** ** ND **UF * ** ** ** ** * ** ND ** UG ** ** ** ** ** ** ** ND ** UH *** *** ****** *** *** ** ND ** UI ** ** ** * ** ** ** ND * UJ ** ** *** * ** * **ND ND UK *** *** *** *** * * ** ND * UL *** ** * ** * ** ** ND * UM **** ** * ** *** ** ND ** UN *** *** ** * ** ** ** ND ND UO ****** * * * * ** ND ** UP * *** ** * ** * ** ND ND UQ * ** ** * *** * **ND ** UR * * ** * ** * ** ND ** US ** ** ** * ** * ** ND ** UT * * * *** * ** ND * UU ** * ** ** ** * ** ND ND UV * *** ** *** * NT ** ND **UW * *** ** ** ** NT ** ND ** UX * *** ** *** ** NT ** ND * UY * ***** * ** NT ** ND ** UZ * *** ** ** * NT ** ND ** VA * * ** * * * ** **** VB * * ** * * * ** ** ** VC * * ** * * * ** ** ** VD NT * ** * * * **** ** VE * * * NT * NT ** ** * VF * * * NT * NT ** ** * VG * * ** NT *NT ** * ND VH NT ** NT ** NT *** ** ** ** VI NT * NT * NT * ** * * VJNT * NT ** NT * ** ** ** VK NT * NT ** NT ** ** ** ** VL * NT * ** * ***** ND * VM * NT ** * * * ** ND * VN ** NT * ** * * ** ND * VO NT * NT*** NT * ** ** * VP *** *** *** NT *** NT ** ND ND VQ * ** * NT ** NT **ND ND VR * * ** NT ** NT ** ND ND VS ** * ** * ** ** ** ND ** VT * *** * *** *** ** ND * VU *** *** *** *** *** *** ** ND ** VV * * ** * ***** ** ND * VW * * ** * ** * ** ND ** VX * * * * ** ** ** ND ** VY ** **** * ** * ** ND ND VZ ** * ** * ** * ** ND ** WA ** ** *** * ** * ** ND** WB *** ** *** *** ** * ** ND ** WC * * *** ** ** ** ** ND ** WD ***** ** * ** * ** ND ** WE *** ** *** ** *** * ** ND ND WF ** ** *** ***** ** ** ND ** WG * * *** * ** * ** ND ND WH * ** ** * ** ** ** ND **WI * * *** ** ** *** ** ND ** WJ * * ** * ** * ** ND * WK *** * *** **** * ** ND * WL ** * ** * ** * ** ND ** WM ** * * * ** ** ** ND * WN** * * * * * ** ND * WO ** * * ** ** *** ** ND ** WP ** * * * ** ** **ND * WQ ** * * *** *** *** ** ND ND WR * * * * ** * ** ND ND WS*** * * * * * ** ND ND WT ** ** * * * * ** ND ** WU ** * * * ** * **ND * WV *** * * * *** * ** ND ** WW *** * * * *** * ** ND * WX ***** * * ** * ** ND * WY *** * * * *** * ** ND ** WZ *** * * * *** * ** NDND XA *** * * * *** * ** ND ND XB *** * * * *** * ** ND * XC *** * * **** * ** ND ND XD ** *** * * * * ** ND ND XE *** * ** * *** ** ** ND NDXF NT *** NT *** NT ** ** * ND XG NT * NT ** NT * ** * * XH NT * NT **NT * ** ** ** XI NT ** NT ** NT *** ** * ** XG NT * NT ** NT * ** ** *XH NT * NT * NT * ** * ND XI NT * NT * NT * ** ** * XJ NT * NT ** NT ***** ** ** XK NT * NT * NT * ** * * XL ** ** ** ** ** ** ** ND * XM * ***** * * * ** ND ** XN ** ** * * * * ** ND ND XO *** *** * *** * * ** ND** XP * ** ** * ** ** ** ND ND XQ *** ** ** * ** ** ** ND * XR ** ***** * ** * ** ND ** XS * ** ** * ** * ** ND ** XT * ** ** * ** * ** ND **XU * * ** * ** * ** ND * XV ** ** * * ** * ** ND * XW * ** ** * ** ** **ND ND XY * * ** *** ** * ** ND ** XZ * *** ** *** * NT ** ND ** YA ** **** * * * ** ** * YB * * ** * * *** ** ** ** YC * * ** * * ** ** ** ** YD** * *** NT * NT ** ** ** YE ** * ** NT * NT ** ** * YF NT *** NT *** NT*** ** ** ** YG NT * NT ** NT *** ** ** * YH NT *** NT *** NT * ** ** **YI NT *** NT *** NT * ** ** ** YJ * NT * ** * * ** ND ** YK * NT ****** * * ** ND * YL * NT * ** * * ** ND ** YM * NT ** ** * * ** ND * YN *NT * ** * ** ** ND ** YO NT * NT * NT * ** ** ND YP NT * NT * NT * ** **** YQ NT * NT ** NT * ** ** ** YR * ** ** NT *** NT ** ND ND YS *** **** * *** *** ** ND * YT ** *** ** * *** * ** ND * YU ** ** ** * ** ** **ND ** YV ** * *** * *** * ** ND ** YW *** *** *** *** *** *** ** ND **YX ** * ** * ** ** ** ND ** YY ** * ** * ** * ** ND ** YZ ** ** *** **** * ** ND * ZA ** * ** * ** * ** ND ** ZB * * *** * ** * ** ND ND ZC** *** *** *** ** *** ** ND ** ZD *** *** *** *** *** *** ** ND ND ZE **** *** * *** *** ** ND ND ZF *** ** ** * *** * ** ND * ZG ** ** ** * ****** ** ND ND ZH ** * * ** * *** ** ND * ZI *** * * * *** * ** ND ND ZJ*** * ** * *** * ** ND * ZK NT * NT * NT * ** * ND ZL NT * NT * NT *** * ND ZM NT * NT ** NT ** ** * ND ZN NT * NT ** NT * ** * ND ZO NT *NT * NT * ** * ** ZP NT * NT ** NT *** ** ** ** ZQ NT ** NT *** NT ***** * ND ZR NT *** NT *** NT * ** ** ** ZS *** * *** *** ** * ** ND **ZT * ** ** * ** ** ** ND ** ZU ** * * * * * ** ND ** AAA * * ** * ** *** ND * AAB *** *** *** *** *** * ** ND ND AAC ** ** ** * ** * ** ND NDAAD * *** ** * ** * ** ND * AAE *** ** ** * ** * ** ND ** AAF ***** * * * * ** ND * AAG * *** ** * ** * ** ND ** AAH *** ** ** * ** * **ND ** AAI * ** ** * ** *** ** ND ND AAJ * *** ** ** * NT ** ND ND AAK **** ** * ** NT ** ND * AAL * * ** * ** * ** ND ND AAM * ** *** *** * **** ** ND AAN * * ** * * * ** ** ND AAO * * ** * * * ** * * AAP * *** * * * ** * * AAQ * ** ** * ** * ** ** ** AAR * * ** * * * ** ** **AAS * * ** ** * *** ** ** * AAT * * ** *** * ** ** ** * AAU * NT * * * *** ND * AAV * NT *** * * *** ** ND ** AAW * NT * *** * ** ** ND ND AAX*** NT ** *** * ** ** ND ** AAZ * NT ** * * * ** ND ND ABA * NT ** **** * ** ND ** ABB * NT * *** * *** ** ND ** ABC NT *** NT *** NT * ** **** ABD NT * NT * NT * ** * ND ABE NT * NT ** NT * ** * ** ABF NT * NT *NT * ** ** ND ABG NT * NT ** NT *** ** * ND ABH NT * NT * NT * ** ** *ABI * ** * NT * NT ** ND ** ABJ ** * *** * ** *** ** ND * ABK ** * *** *** ** ** ND ** ABL ** * ** * ** * ** ND ** ABN ** * ** * ** * ** ND **ABO * * ** * ** *** ** ND ** ABP *** *** *** * ** * ** ND ** ABQ * *** * ** * ** ND ** ABR * * *** * ** * ** ND ** ABS * * *** * ** * ** ND** ABT ** * ** * ** * ** ND * ABU * * ** * *** * ** ND ** ABV * * ** ***** *** ** ND ** ABW * * * * * * ** ND ** ABX *** *** *** *** ** ** **ND ** ABY ** * * * * * ** ND ** ABZ ** * * ** ** *** ** ND * ACA *** *** ** *** *** ** ND * ACB * * * * * * ** ND ND ACC *** * ** * ** * ** NDND ACD *** * * * * * ** ND * ACE ** * * * * * ** ND ND ACF *** * * **** * ** ND * ACG *** * * * ** * ** ND ** ACH *** ** *** * *** * ** NDND ACI NT * NT ** NT *** ** * ** ACJ NT * NT ** NT * ** * ND ACK NT *NT * NT * ** * ND ACL NT * NT * NT * ** * ND ACM *** *** *** *** *** ***** ND ** CAN NT *** NT *** NT ** ** * ** ACO NT *** NT *** NT * ** * **ACP NT *** NT *** NT ** ** ** ** ACQ NT ** NT *** NT ** ** ** ** ACR NT*** NT *** NT * ** ** ** ACS ** * ** * ** * ** ND ** ACT ** ** ** * ***** ** ND ** ACU ** ** ** * *** *** ** ND ** ACV *** * *** ** *** * ** ND** ACW ** ** * ** ** ** ** ND * ACX * ** ** ** ** ** ** ND * ACY **** * * ** ** ** ND ND ACZ ** ** * * * * ** ND * ADA ** *** * *** * ***** ND ND ADB * ** ** * ** *** ** ND ND ADC ** *** ** ** ** *** ** ND *ADD *** *** ** *** ** *** ** ND ** ADE *** ** ** * ** * ** ND * ADF * **** * ** * ** ND ** ADG * * ** * ** * ** ND ** ADH ** ** ** * ** * ** NDND ADI *** ** * * * * ** ND * ADJ * ** ** * ** * ** ND ** ADK * ** ** *** * ** ND ** ADL * * ** * ** * ** ND ** ADM * ** ** * ** * ** ND * ADN** ** ** * ** * ** ND * ADO *** ** ** * ** * ** ND ND ADP * ** ** ** ***** ** ND * ADQ ** ** ** *** ** * ** ND ND ADR * *** ** ** ** NT ** ND** ADS * * ** ** * ** * ** ** ADT * * ** * * * * ** ** ADU * NT * * *** * * ** ADV * NT ** ** * * * * ND ADW * NT ** * * * * * ND ADX NT ***NT *** NT * * * *

TABLE 5 Mer A Mer A

ID STRUCTURE IGEN Lance IGEN Lance IGEN Lance CO CV Lu

ADY

NT * NT * NT * * ** ** ADZ

NT * NT ** NT ** * ** ** AEA

** * *** * *** * * * ** AEB

** * ** * ** *** * * ** AEC

* * *** * ** ** * * ** AED

** *** *** * ** * * * ** AEF

** * ** * ** * * * ND EG

** * *** ** *** *** * * ** AEH

** * *** * ** * * * ** AEI

* * * * * * * * * AEJ

** * * ** ** *** * * * AEK

*** * * ** *** ** * * ND AEL

NT * NT * NT * * * ND AEM

NT * NT * NT ** * * ** EN

NT * NT * NT * * ** ND AEO

NT * NT * NT * * * ** AEQ

NT * NT * NT * * ** ** ER

NT * NT *** NT *** * ** ** AES

*** *** *** * *** * * * ** AET

NT *** NT *** NT * * * ** EU

NT *** NT *** NT * * ** ** AEV

* ** ** ** ** * * * * AEW

** ** ** * ** * * * * EX

* ** ** ** ** * * * ND AEY

** ** ** ** ** ** * * ** AEZ

* ** * * * * * * ** AFA

** ** ** * ** ** * * * FB

* ** ** * ** ** * * ** AFC

** ** ** * ** * * * ** AFD

*** * *** * ** * * * ** AFE

** * ** * ** * * * ** AFG

* ** ** * ** * * * ** AFH

* * ** * ** * * * ND AFI

* ** *** ** ** * * * ** AFJ

* * ** ** * ** * ** * AFK

* * ** * * ** * ** ** AFL

** * ** * ** * * ** * AFM

NT * *** * ** * * ** ** AFN

* * * NT * NT * ** * AFO

NT * NT * NT *** * ** ND AFP

NT * NT * NT * * ** ** AFQ

NT * * NT * * ** ** AFR

*** NT *** *** * *** * * * AFS

** NT *** ** ** ** * * * AFT

* NT * * * * * * * AFU

* * * NT * NT * * ND AFV

** * ** * ** *** * * ** AFW

** * ** * ** * * * * AFX

** * *** * ** * * * ** AFY

*** *** *** *** ** * * * ND AFZ

* * ** * ** *** * * ** AGA

* * * * * * * * ND AGB

*** * * ** *** *** * * * AGC

** * * ** *** *** * * * AGD

** * * * * * * * ND AGE

NT ** NT ** NT *** * * ** AGF

NT * NT * NT * * * ** AGG

NT * NT ** NT ** * * ** AGH

NT * NT * NT ** * * ** AGI

NT * NT * NT * * * ** AGJ

** * *** * ** * * * ** AGK

NT * NT ** NT * * ** ** AGL

NT *** NT *** NT * * * ** AGM

NT *** NT *** NT ** * * ** AGN

* ** ** * ** ** * * * AGO

** ** ** * ** * * * * AGP

** ** *** ** ** ** * * ** AGQ

** ** ** * ** * * * ** AGR

*** *** ** * ** ** * * ND AGS

* *** ** * ** ** * * ** AGT

* * ** * ** * * * * AGU

** ** ** * ** * * * ND AGV

* *** ** *** ** ** * * ** AGW

* *** ** ** ** NT * * * AGX

NT ** NT ** NT *** * ** ** AGY

NT * NT ** NT * * ** * AGZ

NT *** NT *** NT * * ** ** AHA

NT * NT ** NT * * ** ** AHB

* NT * ** * * * * ** AHC

NT * NT ** NT * * * * AHD

NT * NT * NT * * ** * AHE

*** * *** * ** * * * * AHF

*** * ** * ** * * * ** AHG

** * ** * ** * * * ** AHH

* * *** * ** * * * ** AHI

* * * * *** * * * ND AHJ

** * ** * *** * * * ND HK

** ** * ** ** *** * * ** AHL

NT * NT * NT * * * ND AHM

NT * NT * NT * * * ND AHN

NT ** NT ** NT *** * * ND AHO

NT * NT * NT * * * ND AHP

NT *** NT *** NT ** * ** ** AHQ

** * * * * * * * * AHR

*** *** *** *** *** *** * * * AHS

* ** ** * ** ** * * * AHT

* ** ** * ** * * * ** AHU

* ** ** * ** * * * ND AHV

* ** ** * ** * * * ** AHW

** ** ** *** ** ** * * ND AHX

* *** ** *** ** NT * * ND AHY

* * ** ** * *** * ** ** AHZ

* * ** NT * NT * ** ND AIA

NT ** NT ** NT * * ** ND AIB

** * *** * ** * * * ** AIC

** *** *** *** ** *** * * ** AID

** * ** * *** * * * ND AIE

** * * * * * * * * AIF

*** * * * *** * * * ND AIG

** ** * * * * * * ND AIH

NT * NT * NT * * * ** AII

NT * NT ** NT * * * ND AIJ

NT *** NT *** NT * * ** ND AIK

*** ** *** ** ** * * * ND AIL

** *** * ** * *** * * * AIM

* * ** * ** * * * ** AIN

** * ** * ** * * * ** AIO

* * ** ** ** ** * * ** AIP

* ** ** * ** * * * ND AIQ

* ** ** ** ** ** * * ND AIR

** * ** * ** * * ** ** AIS

* * ** * ** * * ** ** AIT

* * NT * * * * ** ** AIU

* * ** NT * NT * * * AIV

NT *** NT *** NT ** * ** ** AIW

*** NT *** *** ** * * * ** AIX

** * ** * ** * * * ** AIY

** * ** * *** * * * ** AIZ

* * *** * *** * * * ND AJA

* * ** * ** * * * ND AJB

* * *** * ** * * * ND AJD

** ** * * ** * * * * AJE

NT * NT ** NT * * * ND AJF

NT * NT * NT * * * * AJG

NT *** NT *** NT *** * * ** AJH

** *** *** * ** * * * ** AJI

** ** * * * ** * * * AJJ

** ** * ** * * * * * AJK

*** * ** * ** * * * ND AJL

* ** ** * ** * * * * AJM

* ** ** ** ** *** * * ND AJN

* *** ** ** ** NT * * ** AJO

* NT ** * ** NT * * ** AJP

* *** ** * ** NT * * * PJQ

NT * NT * NT * * * * AJR

NT * NT ** NT *** * * ** AJZ

* * ** NT ** NT * * ** AKA

** * ** * ** *** * * ** AKB

** ** *** ** ** *** * * ** AKC

** * ** ** *** *** * * ND AKD

** * * * * * * * ND AKE

*** * * ** *** *** * * ** AKF

*** * * ** *** *** * * * AKG

*** * * * ** ** * * ND AKH

NT *** NT *** NT *** * * ** AKI

* * ** ** ** *** * * ** AKJ

* NT ** ** * * * * ND AKK

* NT ** ** * ** * * ** AKL

NT * NT ** NT * * * * AKM

NT * NT *** NT *** * ** ** AKN

NT ** NT ** NT * * ** * AKO

* * ** * ** *** * * ** AKP

* NT ** ** ** *** * * ND AKQ

NT ** NT ** NT * * * ** AKR

*** *** ** * ** * * * * AKS

*** *** ** * ** ** * * ** AKT

** ** *** *** *** *** * * ** AKU

NT ** NT *** NT *** * * ** AKV

NT ** NT ** NT ** * ** * AKW

NT *** NT *** NT * * ** ** AKX

NT *** NT *** NT *** * ** ** AKY

* * ** * ** * * ** ** AKZ

NT ** NT ** NT ** * ** ** ALA

NT * NT * NT * * * ** ALB

* NT ** * * * * * ND ALC

** ** ** * *** * * * ** ALD

* ** ** NT ** NT * * ND ALE

* * ** * ** * * * ** ALF

* ** * ** * *** * * ND ALG

* *** * ** * *** * * * ALH

* ** * * * * * * ALI

** ** * * * * * * **

ALJ

*** ** * * *** * * * **

ALK

* NT * ** * *** * * ** ALL

** * * *** * *** * * * ALM

*** ** ** * ** * * * ** ALN

NT * NT ** NT * * ** ** ALO

*** * * *** *** *** * * * ALP

** ** * * * * * * * ALQ

*** *** *** *** *** *** * * * ALR

NT * NT ** NT * * * * ALS

* * * NT * NT * * ** ALT

* *** * ** ** NT * * ** ALU

* * ** ** *** *** * ** ND ALV

*** ** * ** *** *** * ** ND ALW

** * * * * * * ** ND ALX

*** ** *** *** ** * * ** * ALY

NT ** *** * ** * * ** ** ALZ

NT * *** * ** * * ** ** AMA

* * * NT ** NT * ** * AMB

** *** *** *** *** ** * ** ** AMC

* * ** ** ** ** * ** ** AMD

** * ** * ** * * ** * AME

** * * * ** * * ** * AMF

* * * * * * * ** ** AMG

*** *** *** *** *** *** * ** ND AMH

* * * NT ** NT * ** ** AMI

** * ** * ** * * ** ** AMJ

* * ** * * *** * ** ND AMK

NT ** NT *** NT * * ** ** AML

** * ** * *** * * ** ND AMM

** ** * * * ** * ** ND AMN

NT * NT ** NT * * * * AMO

* * ** * * ** * ** ** AMP

** * * * * ** * ** ND AMQ

* * ** ** ** * * ** ND AMR

* * * NT ** NT * * * AMS

*** ** * * *** ** * ** ND AMT

* * ** ** ** ** * * ** AMU

*** *** *** *** *** * * ** ** AMV

* * ** * ** * * * * AMX

* NT ** *** * *** * ** ** AMY

** *** *** * ** *** * ** ** AMZ

*** * * * * * * ** ND ANA

*** * ** * *** * * ** ** ANB

** * * * * ** * ** ** ANC

NT * NT ** * ** * ** ** AND

* NT * *** * *** * ** ** ANE

*** * *** * ** * * ** ** ANF

*** *** *** *** ** *** * ** ** ANG

** * ** * ** * * ** ** ANH

* ** ** *** * NT * ** * ANI

NT NT NT NT NT NT NT NT NT ANJ

NT NT NT NT NT NT NT NT NT ANK

NT NT NT NT NT NT NT NT NT ANL

NT NT NT NT NT NT NT NT NT ANM

NT NT NT NT NT NT NT NT NT ANN

NT NT NT NT NT NT NT NT NT ANO

NT NT NT NT NT NT NT NT NT ANP

NT * NT NT * NT NT NT NT ANQ

NT * NT NT * NT NT NT NT ANR

NT * NT NT * NT NT NT NT ANS

NT * NT NT * NT NT NT NT ANT

NT * NT NT * NT NT NT NT

Example 9 Luciferase Assay

The luciferase assay is used to determine if any of the compounds testedreduce the luminescent signal. This indicates that the test compoundsaffect regulation of micF, which in turn is regulated by Mar.

Materials

The bacteria used were E. coli AG 112 KmicF-Luc. The negative controlBacteria were E. coli AG112. The test compounds were prepared in a 10mg/mL DMSO stock solution.

Methods

Preparation of Inoculum

Inoculum (or “Starter Inoculum”) was started the night before the day ofthe experiment by adding either a colony or stab of a glycerol stock to2 mL of LB Broth. The Starter Inoculum was then placed in a 37° C.shaker incubator and allowed to grow overnight.

The following day, the Starter Inoculum was removed from the shaker andadded to fresh LB Broth. For each plate to be assayed, 6 mL of LB brothwas prepared, with 5-10 μL of Starter Inoculum being added per mL ofadded LB to form the “Test Inoculum”. Typically, four plates of testcompounds were assayed. In this typical example, 6 mL of LB Broth wasused for each plate, or 24 mL of LB, followed by the addition of 5 mL/mLof Starter Inoculum, or 120 μL of Starter Inoculum to form the TestInoculum.

Following preparation of the Test Inoculum, the Test Inoculum was placedin a 37 degree Celsius shaker and incubated for about 4 hours. The TestInoculum was monitored for bacterial growth by taking OD readings at 535nm on a spectrophotometer. The Test inoculum should be removed when theOD reaches between 0.6 and 1.5.

Preparation of Controls

Positive and negative controls were created by adding 2 uL DMSO to 198uL LB Broth. At least 4 of each control were generated. Typically, therewere 8 of each. 50 uL of diluted DMSO was added to 50 uL LB Broth in theassay plates.

Preparation of Compounds

The compounds were screened at 25 ug/mL. Two identical plates of eachcompound were set up: 1 clear plate for growth (or “Clear Plate”), 1white plate for luminescence (or “White Plate”). Next, 2 μL of eachcompound was taken from the daughter plate (containing 10 mg/mL stock)and added to 198 μL of LB Broth. The sample was then mixed. Next, 25 μLof the diluted test compound was added to 25 μL of LB Broth in all ofthe assay plates. The concentration of the compound at this stage was 50μg/mL.

Preparation of Plate

50 μL of the Test Inoculum was added to each well of the plates, exceptfor the negative controls. Half of the negative controls received 50 μLof AG112, while the other half of the negative controls received 50 μLLB Broth. The final concentration of the test compound was 25 μg/mL.

The Clear Plates were placed in the plate reader and read at OD₅₃₅. Thiswas the “Initial” growth read. The plates were then incubated plates for5 hours at 37 degrees Celsius. After 5 hours, the plates were removedfrom the incubator. The Clear Plates were placed in the plate reader andread at OD₅₃₅. This was the “Final” growth read.

100 μL of Promega Steady-Glo reagent was added to each well (includingall controls) in the White Plates. The plates, covered with aluminumfoil, were then shaken on a plate shaker set at 10000 rpm for 10 min.The plates were then placed in plate reader and read on luminescence for1 sec per well. This was the LUMINESCENT read.

Data Analysis

To determine whether the test compound inhibited growth, the Initialgrowth read was subtracted from the Final growth read. This was theSubtracted Growth. The same calculation was performed for the positivecontrols. The results for the positive controls were averaged. The %Inhibition of Growth was determined using the following formula:100−(100*Subtracted growth of sample/Average growth of Pos Controls)To determine whether compound inhibits Luciferase, use the followingequation:100−(100*Luminescence for Compound/Average Luminescence of Pos Controls)

ND indicates that a particular test compound did not appear to decreasethe lumninesce of in this particular assay. * indicates that theluminescence was decreased somewhat and ** indicates that theluminescence was decreased a substantial amount. The results from thisassay are also shown in Table 5.

Example 10 Synthesis of Various Benzimidazole Compounds

(2,4-Dinitro-phenylamino)-acetic acid methyl ester (2). A mixture of1-fluoro-2,4-dinitrobenzene (1) (15 g, 0.81 mmol), glycine methyl esterhydrochloride (11.5 g, 0.92 mmol), K₂CO₃ (22.3 g, 0.162 mmol) andmethanol (300 mL) was heated at 60° C. for 30 minutes. After cooling inan ice bath, the resulting yellow precipitate was collected byfiltration, washed with water and methanol and dried in vacuo. Yield10.5 g (51%).

1-Hydroxy-6-nitro-1H-benzoimidazole-2-carboxylic acid ethyl ester (3). Asolution of (2,4-Dinitro-phenylamino)-acetic acid methyl ester (2) (3 g,11.8 mmol) in ethanol (100 mL) was heated to 70° C. After addition of2.4 mL (24.2 mmol) of piperidine, the solution was refluxed at 70° C.After 2 hours, the solvent was removed in vacuo and the resultingresidue was dissolved in water (100 mL). Acidification of the solutionwith HCl yielded a yellow precipitate, which was collected byfiltration, washed with water and ethanol and dried in vacuo. Yield 1.9g, (63%) of yellow solid.

6-Nitro-benzoimidazol-1-ol hydrochloride (4). A mixture of1-hydroxy-6-nitro-1H-benzoimidazole-2-carboxylic acid ethyl ester (3) (5g, 20 mmol) and concentrated HCl (100 mL) was refluxed for 3 hours.After cooling the mixture to room temperature, the resulting solid wascollected by filtration. Yield 1.9 g (44%) of the HCl salt.

6-Nitro-benzoimidazol-1-yloxy)-acetic acid ethyl ester (5) To a mixtureof 6-nitro-benzoimidazol-1-ol hydrochloride (4) (2 g, 9.3 mmol) andK₂CO₃ (2.56 g, 19 mmol) in DMF (60 mL) was added ethyl bromoacetate (3.1g, 19 mmol) with stirring at room temperature. After 4 hours, thereaction mixture was poured into water. The resulting solid wascollected by filtration, washed with water and ethanol and dried invacuo. Yield 1.2 g (49%).

(6-Nitro-benzoimidazol-1-yloxy)-acetic acid (6) A mixture of6-nitro-benzoimidazol-1-yloxy)-acetic acid ethyl ester (5) (250 mg, 0.94mmol), THF (5 mL), water (1 mL) and concentrated HCl (1 mL) was heatedto reflux for 2 hours. The reaction mixture was evaporated and the cruderesidue was purified by HPLC (21.2×250 mm Phenomenex Luna C18(2) column;flow rate=20 mL/min; linear gradient 0-100% B over 30 minutes; ABuffer=water with 0.1% TFA, B Buffer=acetonitrile with 0.1% TFA). HPLCsolvents removed in vacuo to yield yellow solid. Yield 65 mg (29%).Synthesis of 2-aryl-benzimidazoles

-   -   R₁=NO₂, F, Cl, Br, NH₂, NHAc, COMe, COPh, CF₃, COOH, OMe, CN,        CONH₂, ^(t)Bu, COOR, etc.    -   R₂=substituted or unsubstituted phenyl, substituted or        unsubstituted heterocycle (5 or 6 membered rings etc.)

To a solution of R¹-substituted-2-nitrofluorobenzene in DMF or DMSO, wasadded 2 equiv of NaHCO₃. Ca. 1.5-2 equivalents of the correspondingsubstituted benzylamine (e.g., R₂=—H, -Me, —NH₂, —Cl, —OMe, —C(═NH)NH₂etc.) was added slowly to the reaction mixture with vigorous stirring.The reaction was monitored by HPLC/TLC and upon complete consumption ofthe starting material, the reaction mixture was poured into ice waterand the precipitate was filtered, washed with excess water andair-dried. In some cases, upon pouring the reaction mixture in water,treatment with 10% dilute HCl (aq.) was needed to wash away excess saltsand any base. The material thus obtained is usually pure, and can beused for the next step without any further purification. Yields arebetween 50-95%. Purity of each of the compounds was confirmed using ¹HNMR spectroscopy, HPLC, and

-   -   R₁=NO₂, F, Cl, Br, NH₂, NHAc, COMe, COPh, CF₃, COOH, OMe, CN,        CONH₂, ^(t)Bu, COOR, etc.    -   R₂=substituted or unsubstituted phenyl, substituted or        unsubstituted heterocycle (5 or 6 membered rings etc.)

A solution/suspension of the substituted nitro compound (from theprevious step) in methanol or THF or methanol/DMF was warmed to 50° C.,treated with an excess base (NaH, CH₃O⁻Na⁺, aq. NaOH, etc.) and stirred.HPLC monitoring of the reaction mixture indicated the completion (10min-12 h depending upon the substituent) of the reaction. The reactionmixture was poured over ice, treated with dil. HCl to acidic pH, and theresulting precipitate was filtered, washed thoroughly with dil HCl, andfinally with water. In cases, where the product was water soluble, thereaction mixture was quenched with ice-water, evaporated to dryness, andthe product was purified via extraction, washing, or if necessary, viachromatography. All the compounds were characterized using HPLC, MS, and¹H NMR spectroscopy. Yields: 30-90%

-   -   R₁=NO₂, F, Cl, Br, NH₂, NHAc, COMe, COPh, CF₃, COOH, OMe, CN,        CONH₂, ^(t)Bu, COOR, etc.    -   R₂=substituted or unsubstituted phenyl, substituted or        unsubstituted heterocycle (5 or 6 membered rings etc.)

To a DMF solution of the benzimidazole-N-hydroxide (from the previousstep) 1.5-2 equiv of anhydrous Na₂CO₃ was added, followed by theaddition of 1.2-1.8 equiv of bromo-acetic acid. The reaction mixture wasstirred at room temperature and monitored via HPLC/MS. Upon completionof the reaction, the reaction mixture was poured over ice and treatedwith dil. HCl to an acidic pH. In most cases the product crashed out ofthe solution, which was filtered, washed thoroughly with dil. HCl, andwater and air dried. The product thus obtained is usually pure, but whenneeded, it could be recrystallized from DMF/ether or methanol/ether ordichloromethane/hexane solvent systems. In cases, where the product iswater soluble, the quenched reaction mixture was concentrated to avolume where the product started to crash out. The final product waspurified via chromatography in such cases or in cases that the crudematerial is impure. The final product was characterized using HPLC, MS,¹H NMR spectroscopy, and in some representative cases, using CHNanalyses and ¹³C NMR spectroscopy as well.

-   -   Y=substituted or unsubstituted phenyl, substituted or        unsubstituted heterocycle (5 or 6 membered rings), etc.

Preparation of N-(4-aminobenzyl)-2,4-dinitroaniline (7) To a solution of4-aminobenzylamine (25.5 mL, 225 mmol) and powdered NaHCO₃ (94.5 g, 1125mmol) in anhydrous DMF (300 mL) was added 2,4-dinitrofluorbenzene (1)(18.8 mL, 150 mmol) dropwise at room temperature. After 2 hours, thesolution was slowly diluted with water (1000 mL) to precipitate theproduct, which was collected on a fritted funnel while rinsing withwater until the eluent was colorless. The solid was further dried underhigh vacuum to afford 43.0 g as a bright orange solid in 99% yield.

Preparation of 6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazole (8) To asolution of N-(4-aminobenzyl)-2,4-dinitroaniline (7) (21.6 g, 74.9 mmol)in anhydrous EtOH (300 mL) and anhydrous DMF (75 mL) was slowly addedsodium methoxide (30% w/w) (69.1 g, 375 mmol) at room temperature underargon atmosphere, followed by heating to 60° C. for 2 hours. Aftercooling to ambient termperature, the solution was diluted with water(700 mL) and then acidified with saturated citric acid. The resultingprecipitate was collected on a sintered funnel while rinsing with water.The crude product was recrystallized in hot EtOH to afford 18.1 g as abrown solid in 90% yield.

General procedure for the preparation ofN-acyl-6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazoles (9) To asolution of 6-nitro-2-(4-aminophenyl)-1-hydroxybenzimidazole (8) (270mg, 1.00 mmol) in anhydrous pyridine (2.0 mL) was added acid chlorides(2.5 mmol) or the in situ mixed anhydrides at room temperature. (Themixed anhydride was prepared by adding trimethylacetyl chloride (2.5mmol) dropwise to a solution of the carboxylic acid (2.55 mmol) inanhydrous pyridine at 0° C. After 1 hour,6-nitro-2-(−4-aminophenyl)-1-hydroxybenzimidazole was added in oneportion.) After stirring for 2-3 hours at room temperature, the solutionwas diluted with 3M NaOH (6.0 mL) and stirred for another hour. The deepamber solution was diluted with water (100 mL) and then acidified withsaturated citric acid. The resulting precipitate was collected on asintered funnel while rinsing with water. The crude product was furtherpurified by either prepatory HPLC or by recrystallization in hot ethanolor a mixture of hot ethanol and chloroform.

-   -   M=substituted or unsubstituted phenyl, substituted or        unsubstituted heterocycle (5 or 6 membered rings), etc

Preparation of6-nitro-2-(4-phenylethynyl-phenyl)-1-hydroxybenzimidazoles (11) Asolution of 6-nitro-2-(4-bromophenyl)-1-hydroxybenzimidazole (10) (334mg, 1 mmol) in DMF (2 mL) and Et₃N (1 mL) was degassed with argon for 30minutes. Phenylacetylene (408 mg), 4 mmol), CuI (38 mg, 0.2 mmol), andPd(PPh₃)₄ (116 mg, 0.1 mmol) were added. Degassing was continued foranother 5 minutes and the reaction vial was placed in a sand bathpreheated to 100° C. overnight. The reaction was cooled and diluted with50 mL of water and the pH was adjusted to pH 4 with 10% aqueous HCl. Thesolids were filtered and triturated successively with 1,2-dichloroethaneand warm methanol. The resultant yellow solid was further purifed bypassing through a silica gel flash column eluting with EtOAc:Hexanes(1:1). Fractions containing the product were pooled and evaporated toprovide 27 mg of a yellow solid.

-   -   A=F, CF₃, CN, COOH, alkylamine, COCH₃, H, etc.    -   Z=F, alkylamine, etc.

Preparation of 4-phenylamidobenzylamine (13) In a pressure reaction,4-dimethylaminophenylamidobenzonitrile (12) (26 g, 98 mmol) wasdissolved in anhydrous THF (940 mL), and the solution was purged withargon for 2-3 minutes, followed by the addition of 11 mL of uniformlysuspended catalyst (Raney® nickel 2400, suspension in water). Afteraddition of a small amount of methanol to the suspension, the reactorwas pressurized at 55 psi H₂ while stirring vigorously for 2.5 hours.The reaction mixture was filtered over a bed of diatomaceous earth (e.g.Celite®), and washed 3×100 mL of anhydrous THF. The combined filtrateswere evaporated to dryness, and further dried under high vacuum toafford 25.1 g of white solid.

Preparation of 4-[(2-nitro-phenylamino)-methyl]-phenylbenzamide (14) Toa solution of 4-phenylamidobenzylamine (13) (225 mmol) and powderedNaHCO₃ (1125 mmol) in anhydrous DMF (300 mL) was added substituted4-nitrofluorbenzene (150 mmol) dropwise at room temperature. After 2 h,the solution was slowly diluted with water (1000 mL) to precipitate theproduct, which was collected on a fritted funnel while rinsing withwater until the eluent was colorless. The solid was further dried underhigh vacuum to afford the product.

Preparation of 4-(benzimidazol-2yl)-phenylbenzamide (15) To a solutionof 4-[(2-nitro-phenylamino)-methyl]-phenylbenzamide (14) (74.9 mmol) inanhydrous EtOH and anhydrous DMF (75 mL) was slowly added sodiummethoxide (30% w/w) (375 mmol) at room temperature under argonatmosphere, followed by heating to 60° C. for 2 h. After cooling toambient termperature, the solution was diluted with water (700 mL) andthen acidified with saturated citric acid. The resulting precipitate wascollected on a sintered funnel while rinsing with water. The crudeproduct was recrystallized in hot EtOH.

-   -   Z=F, alkylamine, etc.

Preparation of 3-aminobenzyldinitrophenylamine (16) To a solution of3-aminobenzylamine (225 mmol) and powdered NaHCO₃ (1125 mmol) inanhydrous DMF (300 mL) was added 2,4-dinitrofluorbenzene (1) (18.8 mL,150 mmol) dropwise at room temperature. After 2 hours, the solution wasslowly diluted with water (1000 mL) to precipitate the product, whichwas collected on a fritted funnel while rinsing with water until theeluent was colorless. The solid was further dried under high vacuum.

Preparation of 3-aminonitrobenzoimidazolol (17) To a solution of3-aminobenzyldinitrophenylamine (16) (74.9 mmol) in anhydrous EtOH (300mL) and anhydrous DMF (75 mL) was slowly added sodium methoxide (30%w/w) (375 mmol) at room temperature under argon atmosphere, followed byheating to 60° C. for 2 h. After cooling to ambient termperature, thesolution was diluted with water (700 mL) and then acidified withsaturated citric acid. The resulting precipitate was collected on asintered funnel while rinsing with water. The crude product wasrecrystallized in hot EtOH.

Preparation of 4-(benzoimidazolyl)phenylbenzamide (18) To a solution of3-aminonitrobenzoimidazolol (17) (1.00 mmol) in anhydrous pyridine (2.0mL) was added acid chlorides^(a) (2.5 mmol) or the in situ mixedanhydrides at room temperature. (The mixed anhydride was prepated byadding trimethylacetyl chloride (2.5 mmol) dropwise to a solution of thecarboxylic acid (2.55 mmol) in anhydrous pyridine at 0° C., After 1hour, 6-nitro-2-(−4-aminophenyl)-1-hydroxybenzimidazole was added in oneportion.) After stirring for 2-3 h at room temperature, the solution wasdiluted with 3M NaOH (6.0 mL) and stirred for another 1 h. The deepamber solution was diluted with water (100 mL) and then acidified withsaturated citric acid. The resulting precipitate was collected on asintered funnel while rinsing with water. The crude product was furtherpurified by either prepatory HPLC or by recrystallization in hot ethanolor a mixture of hot ethanol and chloroform.

Example 11 SoxS Gel Shift Assay of Test Compounds

The test compounds were diluted in DMSO to the required concentrationand added to the appropriate wells. Protein (SoxS) was added to thewells in EMSA buffer at a concentration that was determined to cause a50% shift of the DNA. The plates were then covered, mixed and shaked for30 minutes at room temperature to allow for compound-protein binding.

Ten μl of DNA mix (2.4 μl 5×EMSA buffer, 0.2 μl poly(dIdC), 1 μl ³³P-DNAprobe, 7.4 μl dH₂O per reaction) was then added to each well. The finalDNA concentrations were approximately 1 nM. The samples were then mixedfor 15 minutes at room temperature which allowed protein-DNA complexesto form.

Electrophoresis was started at approximately 110V and the gels werepre-run for 10-15 minutes. Five μl of gel loading buffer was then addedto each sample and mixed. Fifteen μl of each sample was then loaded ontogel. The gel was electrophoresed at 110V for approximately 2 hours oruntil the bromophenol blue marker approached the bottom of the gel. Thegel was then transfered to Whatman filter paper, covered, and dried at80° C. for approximately 30 minutes. Autoradiography film was exposed tothe gel overnight and developed.

The probe alone well showed a single DNA species (unbound) of anapparent low molecular weight. Controls containing protein showedapproximately 50% of the DNA at a shifted or bound position (apparenthigher mwt) and 50% at the same position as the probe alone (free DNA).Samples containing test compounds showed ratios of bands between thesetwo controls. A compound that completely inhibited protein-DNA bindingappeared to be similar to that of the probe alone. Table 6 shows theresults of this assay. Compounds which showed superior inhibition of DNAbinding are indicated by “***,” compounds which showed very good or goodinhibition of DNA binding are indicated by “**” or “*” respectively.Compounds which showed some inhibition of DNA binding are indicated by“-.” TABLE 6

ID STRUCTURE

ANI

— ANJ

— ANK

— ANL

*** ANM

— ANN

— ANO

* ANP

— ANQ

* ANR

— ANS

— ANT

** ANU

— ANV

* ANW

— ANX

— ANY

— ANZ

— AOA

— AOB

— AOC

— AOD

— AOE

* AOF

— AOG

— AOH

— AOI

— AOJ

* AOK

— AOL

— AOM

— AON

* AOO

* AOP

* AOQ

— AOR

— AOS

* AOT

— AOU

*** AOV

— AOW

*** AOX

*** AOY

*** AOZ

* APA

— APB

— APC

— APD

* APE

— APF

— APG

— APH

— API

— APJ

** APK

— APL

— APM

— APN

* APO

— APP

— APQ

** APR

— APS

— APT

** APU

— APV

*** APW

— APX

* APY

** APZ

— AQA

— AQB

— AQC

— AQD

— AQE

— AQF

— AQG

* AQH

— AQI

— AQJ

*** AQK

*** AQL

*** AQM

— AQN

— AQO

— AQP

— AQQ

*** AQR

— AQS

— AQT

— AQU

** AQV

*** AQW

— AQX

— AQY

— AQZ

— ARA

— ARB

*** ARC

— ARD

*** ARE

— ARF

— ARG

— ARH

— ARI

— ARJ

— ARK

— ARL

— ARM

ARN

— ARO

*** ARP

— ARQ

— ART

— ARU

— ARV

*** ARW

— ARX

— ARY

— ARZ

NT ASA

NT ASB

NT ASC

NT ASD

NT ASE

NT ASF

NT ASG

* ASH

NT ASI

NT ASJ

NT ASK

NT ASL

NT ASM

— ASN

*** ASO

NT ASP

** ASQ

NT ASR

NT ASS

NT AST

NT ASU

NT ASV

NT ASW

NT ASX

NT ASY

NT ASZ

NT ATA

*** ATB

NT ATC

NT ATD

NT ATE

NT ATF

NT ATG

NT ATH

NT ATI

NT ATJ

NT ATK

NT ATL

NT ATM

NT ATN

NT ATO

NT ATP

NT ATQ

NT ATR

NT ATS

NT ATT

NT ATU

NT ATV

NT ATW

NT ATX

NT ATY

NT ATZ

NT AUA

NT AUB

NT AUC

NT AUD

NT AUE

NT AUF

NT AUG

NT AUH

NT AUI

NT AUJ

NT

Example 12 Development of Luminescence Assays

A quantitative chemiluminescence-based assay is being used to measurethe DNA binding activity of various MarA (AraC) family members. Withthis technique, a biotinylated double-stranded DNA molecule (2 nM) isincubated with a MarA (AraC) protein (20 nM) fused to 6-histidine(6-His) residues in a streptavidin coated 96-well microtiter (white)plate (Pierce Biotechnology, Rockford, Ill.). Unbound DNA and proteinare removed by washing and a primary monoclonal anti-6His antibody issubsequently added. A second washing is performed and a secondaryHRP-conjugated antibody is then added to the mixture. Excess antibody isremoved by a third wash step and a chemiluminescence substrate (CellSignaling Technology, Beverly, Mass.) is added to the plate.Luminescence is read immediately using a Victor V plate reader(PerkinElmer Life Sciences, Wellesley, Mass.). Compounds that inhibitthe binding of the protein to the DNA result in a loss of protein fromthe plate at the first wash step and are identified by a reducedluminescence signal. The concentration of compound necessary to reducesignal by 50% (EC₅₀/IC₅₀) can be calculated using serial dilutions ofthe inhibitory compounds. Also, single trancription factor modulatorsthat affect different transcription factors have been identified asshown below: TABLE 7 Activity of selected trancription factor modulatorsagainst disparate MarA (AraC) family members. % Identity EC₅₀ (μM)Host-Protein to MarA ARD ASX ASU ATB ATE E. coli MarA 100 11.7 1.2 SoxS42 8.3 4.9 3 2.7 0.82 Rob 51 28 3.4 4.7 7.4 1.3 S. typhimurium Rma 38 173.5 4.9 1.8 SlyA* ND 11.9 21.7 51 14.3 P. mirabilis PqrA 40 13.6 1.4 P.aeruginosa ExsA 24 15.6 2.5 4 4 1.9*SlyA is a MarR protein and is included as a control, to illustratepreferential binding to MarA family members.

EC₅₀'s for other compounds of the invention for SoxS were alsodetermined. Compounds ANU, AOW, AOX, APJ, AQQ, AQV, ARB, ASS, and ASTwas found to have EC₅₀'s higher than that of ARD.

Example 13 In Vivo Activity of Mar Inhibitors in Pyelonephritis Model ofInfection

Groups of female CD 1 mice (n=6) were diuresed and infected with E. coliUPEC strain C189 via intravesicular inoculation. Subsequently, mice weredosed with a Trancription factor modulator (25 mg/kg), a controlcompound, e.g., SXT (Qualitest Pharmaceuticals, Huntsville, Ala.), orvehicle alone (0 mg/kg), via an oral route of administration at the timeof infection and once a day for 4 days thereafter, to maintain aconstant level of drug in the mice. After a 5-day period of infectionand prior to sacrifice via CO₂/O₂ asphyxiation, a urine sample was takenby gentle compression of the abdomen. Following asphyxiation, thebladder and kidneys were removed aseptically. Urine volumes andindividual organ weights were recorded, the organs were suspended insterile PBS containing 0.025% Triton X-100, and then homogenized. Serial10-fold dilutions of the urine samples and homogenates were plated ontoMcConkey agar plates to determine CFU/ml of urine or CFU/gram of organ.

Efficacy in these experiments were defined as a >9-log decrease inCFU/ml of urine or CFU/g organ. These values are in accord with previousexperiments investigating the treatment of UTI in mice. In Table 8, theresults of the example are shown. Compounds which caused a decrease inthe CFU/g of kidney tissue are indicated with a *. Compounds whichshowed no decrease in CFU/g of kidney issue are indicated with a -. Allof the compounds tested in this assay were previously determined to haveat least some in vitro SoxS activity. TABLE 8 Compound Efficacy CompoundEfficacy AQK * AQU — AQZ * ASN — ARB * ASQ — ARD * AST — ARO * ASU —ARV * ATB — ASR * ATE — ATH * ARY * ASK *

Example 14 In Vitro Activity of Mar Inhibitors Against LcrF (VirF) fromY. pseudotuberculosis

The MarA (AraC) family member LcrF (VirF) was cloned, expressed andpurified from Y. pseudotuberculosis. The purifed protein was used todevelop a cell-free system to monitor DNA-protein interactions in vitro.The activities of Mar inhibitors were surveyed agains LcrF to identifyinhibitory activity and % cytotoxicity in whole cell assays at 50 μg/mL.The results of the example are shown in Table 9. Compounds which showedsuperior inhibition of DNA binding are indicated by “***,” and compoundswhich showed very good or good inhibition of DNA binding are indicatedby “**” or “*” respectively. Compounds which showed a minimumcytotoxicity in whole cell assays at 50 μg/mL are indicated by “***,”and compounds which showed some cytotoxicity or a high cytotoxicity areindicated by “**” or “*” respectively. Table 10 gives EC₅₀ data forselected transcription factor modulaters against LrcF (VirF) from Y.pseudotuberculosis. TABLE 10 Activity of selected trancription factormodulators against MarA (AraC) family member LrcF (VirF) from Y.pseudotuberculosis. EC₅₀ EC₅₀ EC₅₀ Compound (μM) Compound (μM) Compound(μM) ASR 18.3 BBS 29 BBP 7.1 ASU 17.7 BBW >57.3 BCK 0.8 ATB 15.5 BBT27.5 BCL 5.1 ATE 36.5 BBV >57.9 BCO 2.2

TABLE 9 % cytotoxicity in Whole cell assay ID MOLECULAR STRUCTURE EC50for VirF (μM) at 50 μg/ml ASU

*** — ATE

** * BAA

* — BAA

* — BAB

** *** BAC

* — BAD

* — BAE

* — BAF

** *** BAG

* — BAH

* — BAI

* — BAJ

** — BAK

** * BAL

* — BAM

* — BAN

* — BAO

* — BAP

* — BAQ

* — BAR

* — BAS

* — BAT

* — BAU

* * BAV

** ** BAW

* — BAX

* — BAY

* — BAZ

* — BBA

* — BBB

* — BBC

* — BBD

* ** BBE

* — BBF

* — BBG

** — BBH

** — BBI

** — BBJ

** ** BBK

* — BBL

* — BBM

** * BBN

* — BBO

* — BBP

*** *** BBQ

* — BBR

* — BBS

** ** BBT

** * BBU

** — BBV

* — BBW

* — BBX

** — BBX

** — BBY

* — BBZ

* ** BCA

* — BCB

* — BCC

* — BCD

* — BCE

* — BCF

** — BCG

* — BCH

** — BCI

* — BCJ

* — BCK

*** — BCL

*** — BCM

** — BCN

** — BCO

*** — BCP

** — BCQ

** — BCR

*** — BCS

* — BCT

** — BCU

* — BCV

** — BCW

* — BCX

* — BCY

* — BCZ

* — BDA

* — BDB

** — BDC

* — BDD

* — BDE

* —

Example 15 Activity of Mar Inhibitors in Whole Cell Systems

Type III secretion, the process whereby cytotoxic proteins (Yops) aresectreted from a bacterium into a host cell, in pathogenic Yersinia spp.is regulated by LcrF. Wild type Y. pseudotuberculosis are toxic towardJ774 tissue culture cells whereas bacteria bearing a mutation in eitheryopJ (a Yop that inhibits eukaryotic signaling pathways) or IcrF. Thecytotoxicity of wild type Y. pseudotuberculosis was exploited in orderto screen compounds for their ability to penetrate the intact bacterialcell and prevent type III secretion by binding to an inactivating LcrFfunction.

The CytoTox 96® assay kit from Promega was used for this assay. Briefly,J774 macrophages were plated out at 2×10⁴ cells per well in 96-wellplates on the day prior to infection. Yersinia pseudotuberculosis weregrown overnight at 26° C. in 2×YT media and then diluted 1:25 or 1:40the following morning into 2×YT supplemented with 20 mM MgCl₂ and 20 mMsodium oxalate. The cultures were grown for a further 90 min at 26° C.and then shifted to 37° C. for 90 minutes. The temperature shift and thesodium oxalate, which chelates calcium, lead to induction of LcrFexpression. Later experiments also included the YPIIIpIB1ΔJ (YopJmutant) and YPIIIpIB1ΔLcrF (LcrF mutant). YPIIIpIB1ΔJ is a YopJ deletionmutant and any cytotoxicity that is unrelated to YopJ (i.e.lps-mediated) will be seen with this strain. The OD600 was measured andthe culture adjusted to an OD600 of 1.0. This should correspond toapproximately 1.25×10⁹ cells/mL. Dilutions were prepared in DMEM (theJ774 culture media) at different multiplicity of infections (MOIs),assuming J774 cell density of 2×10⁴ . Yersinia pseudotuberculosis wereadded in 10 μl aliquots and cells were incubated at 37° C. either in achamber with a CO₂ generating system, or later, in a tissue cultureincubator with 5% CO₂ for 2 h. Gentamicin was then added to a finalconcentration of 50 μg/ml and the incubations were continued either fora further 2-3 h or overnight. Controls were included for media alone,target cell spontaneous lysis, target cell maximum lysis and effectorcell spontaneous lysis. For maximum lysis, triton X-100 was added to afinal concentration of 0.8% 45 minutes prior to termination of theexperiment. Supernatants containing released LDH were harvestedfollowing centrifugation at 1,000 rpm for 5 minutes. Supernatants wereeither frozen overnight or assayed immediately. 50 μl of supernatant wasmixed with 50 μl fresh assay buffer and incubated in the dark for 30minutes 50 μl of stop solution was added to each well and the plateswere read at 490 nm. The results of this assay are shown in Table 9 andTable 11. Compounds which showed a minimum cytotoxicity in whole cellassays at 50 μg/mL are indicated by “***,” and compounds which showedsome cytotoxicity or high cytotoxicity are indicated by “**” or “*”respectively. TABLE 11 Activity of select Mar Inhibitors in an Assaythat Measures Type III sectretion of Yersinia pseudotuberculosis.Compound % Cytotoxicity AQQ * ASR ** ASU * ATB ** BAB *** BAF ***

Example 16 Investigation of Toxicity of Select Mar Inhibitors In Vivo

The in vivo toxicity of Mar inhibitors ATB, BAB, BAF, BBP and BBS wereinvestigated in repeat dose experiments. Mice were given twosubcutaneous doses (25 mg/Kg) of a Mar inhibitor on day 1 and a singledose of the compound on days 2-5. No overt signs of toxicity for any ofthe compounds were noted indicating that the Mar inhibitors aregenerally well tolerated.

Example 17 Development of a Murine Model of Yersina spp. to MeasureCompound Efficacy

An in-frame and non-polar deletion in lcrF was contructed to measure theeffect of LcrF on the virulence of Y. pseudotuberculosis. Follwinginfection with ˜150 colony forming units (CFU), the LcrF mutantexhibited a >2-log decrease in its ability to colonize the murine lung.When analyzed in a lethal infection model, the 50% lethal dose of wildtype Y. pseudotuberculosis was ˜5-10 CFU whereas the LD50 was >2,220 CFUfor the mutant organism.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims. TABLE 12

TABLE 13

1. A method for reducing antibiotic resistance of a microbial cell,comprising contacting said cell with a transcription factor modulatingcompound of the formula (XI) (XII), (XIII), (XIV), (XV), (XVI), or(XVII):

wherein R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group, or a substituted or unsubstitutedstraight or branched C₁-C₅ alkyl group; R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of H, (C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO₂(C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO(C₁-C₅substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and R^(21a) andR^(21b) are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²² isselected from the group consisting of H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy,heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²³ and R²⁴ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁵ and R²⁶ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁷ is selected fromthe group consisting of substituted heteroaryl; substituted alkyl;substituted or unsubstituted alkenyl; alkynyl; alkylcarbonyl,arylcarbonyl; heteroarylcarbonyl; sulfonyl; alkylamino; arylamino;heteroarylamino; alkoxy, aryloxy, heteroaryloxy; substituted straightchain C₁-C₅ alkyl or alkenyl; substituted or unsubstituted isoxazole,thiazolidine, imidazole, quinoline, pyrrole, triazole, or pyrazine;2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl, and1-fluorophenyl; and R²⁸ is selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²⁹, R³⁰and R³¹ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R³² isselected from the group consisting of OH, Br, CN, COH, morpholinyl,substituted aryl, substituted or unsubstituted alkenyl, alkynyl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, dialkylamino, arylamino, heteroarylamino, andaroyl; R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof; providedthat when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO, then R²³ is notmethyl, unsubstituted phenyl, or unsubstituted furanyl; provided thatwhen R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO, then R²⁵ is notunsubstituted phenyl or O-tert-butyl; provided that when R¹ is OH, R⁴,R⁵, R⁷ and R³³ are H, R⁶ is NO₂, then R³² is not dimethylamino; andprovided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is Br, thenR³² is not dimethylamino; such that the antibiotic resistance of saidcell is reduced.
 2. A method for modulating transcription, comprisingcontacting a transcription factor with a transcription factor modulatingcompound of the formula (XI), (XII), (XIII), (XIV), (XV), (XVI), or(XVII):

wherein R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group, or a substituted or unsubstitutedstraight or branched C₁-C₅ alkyl group; R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of H, (C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO₂(C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO(C₁-C₅substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and R^(21a) andR^(21b) are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²² isselected from the group consisting of H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy,heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²³ and R²⁴ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁵ and R²⁶ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁷ is selected fromthe group consisting of substituted heteroaryl; substituted alkyl;substituted or unsubstituted alkenyl; alkynyl; alkylcarbonyl,arylcarbonyl; heteroarylcarbonyl; sulfonyl: alkylamino; arylamino;heteroarylamino; alkoxy, aryloxy, heteroaryloxy; substituted straightchain C₁-C₅ alkyl or alkenyl; substituted or unsubstituted isoxazole,thiazolidine, imidazole, quinoline, pyrrole, triazole, or pyrazine;2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl, and1-fluorophenyl, and R²⁸ is selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²⁹, R³⁰and R³¹ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R³² isselected from the group consisting of OH, Br, CN, CO₂H, morpholinyl,substituted aryl, substituted or unsubstituted alkenyl, alkynyl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, dialkylamino, arylamino, heteroarylamino, andaroyl; R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof; providedthat when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, then R²³ is notmethyl, unsubstituted phenyl, or unsubstituted furanyl; provided thatwhen R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, then R²⁵ is notunsubstituted phenyl or O-tert-butyl: provided that when R¹ is OH, R⁴,R⁵, R⁷ and R³³ are H, R⁶ is NO₂, then R³² is not dimethylamino; andprovided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is Br, thenR³² is not dimethylamino; such that the transcription is modulated.3.-8. (canceled)
 9. The method of claim 1, wherein R¹ is OH.
 10. Themethod claim 1, wherein R⁴, R⁵, R⁷ and R²⁶ are each H.
 11. The method ofclaim 1, wherein R⁶ is NO₂.
 12. The method of claim 1, wherein R²⁵ is asubstituted alkenyl group.
 13. The method of claim 12, wherein saidsubstituted alkenyl group is substituted with substituted orunsubstituted phenyl.
 14. The method of claim 13, wherein saidsubstituted phenyl is para-halogenated phenyl.
 15. The method of claim14, wherein said substituted phenyl is para-fluorophenyl. 16-77.(canceled)
 78. The method of claim 1, wherein the transcription factormodulating compound is:


79. The method of claim 1, wherein said transcription factor is atranscriptional activation factor.
 80. The method of claim 79, whereinsaid transcriptional activation factor is an AraC family polypeptide.81. The method of claim 79, wherein said transcriptional activationfactor is a MarA family polypeptide.
 82. The method of claim 1, whereinsaid transcription factor modulating compound is a transcription factorinhibiting compound.
 83. The method of claim 1, wherein saidtranscription factor is prokaryotic.
 84. The method of claim 81, whereinsaid MarA family polypeptide is MarA, SoxS, Rob or LcrF (VirF).
 85. Themethod of claim 1, wherein said transcription factor modulating compoundhas an EC₅₀ activity against SoxS of less than 10 μM. 86-96. (canceled)97. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a transcription factor modulating compound,wherein said compound is of the formula (XI), (XII), (XIII), (XIV),(XV), (XVI), or (XVII):

wherein R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group, or a substituted or unsubstitutedstraight or branched C₁-C₅ alkyl group; R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of H, (C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO₂(C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO(C₁-C₅substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and R^(21a) andR^(21b) are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²² isselected from the group consisting of H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy,heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²³ and R²⁴ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁵ and R²⁶ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁷ is selected fromthe group consisting of substituted heteroaryl; substituted alkyl;substituted or unsubstituted alkenyl; alkynyl; alkylcarbonyl,arylcarbonyl; heteroarylcarbonyl; sulfonyl; alkylamino; arylamino;heteroarylamino; alkoxy, aryloxy, heteroaryloxy; substituted straightchain C₁-C₅ alkyl or alkenyl; substituted or unsubstituted isoxazole,thiazolidine, imidazole, quinoline, pyrrole, triazole, or pyrazine;2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl, and1-fluorophenyl; and R²⁸ is selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²⁹, R³⁰and R³¹ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R³² isselected from the group consisting of OH, Br, CN, CO₂H, morpholinyl,substituted aryl, substituted or unsubstituted alkenyl, alkynyl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl;R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, and aroyl;provided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is NO₂, thenR³² is not dimethylamino; provided that when R¹ is OH, R⁴, R⁵, R⁷ andR³³ are H, R⁶ is Br, then R³² is not dimethylamino; provided that whenR¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, then R²⁵ is notunsubstituted phenyl or O-tert-butyl; provided that when R¹ is OH, R⁴,R⁵, and R⁷ are H, and R⁶ is NO₂, then R²³ is not methyl, unsubstitutedphenyl, or unsubstituted furanyl, or a pharmaceutically acceptable salt,ester or prodrug thereof. 98-109. (canceled)
 110. A method forpreventing a bacterial associated state in a subject, comprisingadministering to said subject an effective amount of a transcriptionfactor modulating compound of the formula (XI), (XII), (XIII), (XIV),(XV), (XVI), or (XVII):

wherein R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group, or a substituted or unsubstitutedstraight or branched C₁-C₅ alkyl group; R⁴, R⁵, R⁶, and R⁷ arcindependently selected from the group consisting of H, (C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO₂(C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO(C₁-C₅substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and R^(21a) andR^(21b) are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²² isselected from the group consisting of H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy,heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²³ and R²⁴ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁵ and R²⁶ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁷ is selected fromthe group consisting of substituted heteroaryl; substituted alkyl;substituted or unsubstituted alkenyl; alkynyl; alkylcarbonyl,arylcarbonyl; heteroarylcarbonyl; sulfonyl; alkylamino; arylamino;heteroarylamino; alkoxy, aryloxy, heteroaryloxy; substituted straightchain C₁-C₅ alkyl or alkenyl; substituted or unsubstituted isoxazole,thiazolidine, imidazole, quinoline, pyrrole, triazole, or pyrazine;2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl, and1-fluorophenyl; and R²⁸ is selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²⁹, R³⁰and R³¹ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R³² isselected from the group consisting of OH, Br, CN, CO₂H, morpholinyl,substituted aryl, substituted or unsubstituted alkenyl, alkynyl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl;R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, and aroyl;provided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is NO₂, thenR³² is not dimethylamino; provided that when R¹ is OH, R⁴, R⁵, R⁷ andR³³ are H, R⁶ is Br, then R³² is not dimethylamino; provided that whenR¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, then R²⁵ is notunsubstituted phenyl or O-tert-butyl; provided that when R¹ is OH, R⁴,R⁵, and R⁷ are H, and R⁶ is NO₂, then R²³ is not methyl, unsubstitutedphenyl, or unsubstituted furanyl, or a pharmaceutically acceptable salt,ester or prodrug thereof, such that the bacterial associated state insaid subject is prevented.
 111. The method of claim 110, wherein saidsubject is a human. 112-115. (canceled)
 116. A compound of the formula(XI), (XII) (XIII), (XIV), (XV), (XVI), or (XVII):

wherein R¹ is OH, OCOCO₂H, a substituted or unsubstituted straight orbranched C₁-C₅ alkyloxy group, or a substituted or unsubstitutedstraight or branched C₁-C₅ alkyl group; R⁴, R⁵, R⁶, and R⁷ areindependently selected from the group consisting of H, (C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO₂(C₁-C₅substituted or unsubstituted, straight or branched alkyl), CO(C₁-C₅substituted or unsubstituted, straight or branched alkyl),CO(substituted or unsubstituted aryl or heteroaryl), CO(C₃-C₆substituted or unsubstituted cycloalkyl), O(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), C(NOH)(C₁-C₅ substituted orunsubstituted, straight or branched alkyl), substituted or unsubstitutedamino, CO₂H, CN, NO₂, CONH₂, (CO)(NHOH), and halogen; and R^(21a) andR^(21b) are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl; R²² isselected from the group consisting of H, substituted or unsubstitutedalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy, aryloxy,heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²³ and R²⁴ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁵ and R²⁶ areindependently selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, arylamino, heteroarylamino, and aroyl; R²⁷ is selected fromthe group consisting of substituted heteroaryl; substituted alkyl;substituted or unsubstituted alkenyl; alkynyl; alkylcarbonyl,arylcarbonyl; heteroarylcarbonyl; sulfonyl; alkylamino; arylamino;heteroarylamino; alkoxy, aryloxy heteroaryloxy; substituted straightchain C₁-C₅ alkyl or alkenyl; substituted or unsubstituted isoxazole,thiazolidine, imidazole, quinoline, pyrrole, triazole, or pyrazine,2-fluorophenyl, 2-methylphenyl, 2-cyanophenyl, 1-methylphenyl, and1-fluorophenyl, and R²⁸ is selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl, R²⁹, R³⁰and R³¹ are independently selected from the group consisting of H,substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl,alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, arylamino, heteroarylamino, and aroyl, R³² isselected from the group consisting of OH, Br, CN, COH, morpholinyl,substituted aryl, substituted or unsubstituted alkenyl, alkynyl,heteroaryl, alkoxy, aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl,aminosulfonyl, alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl,acylamino, alkylamino, dialkylamino, arylamino, heteroarylamino, andaroyl, R³³ is selected from the group consisting of H, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkoxy,aryloxy, heteroaryloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl,alkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, acyl, acylamino,alkylamino, dialkylamino, arylamino, heteroarylamino, aroyl andpharmaceutically acceptable salts, esters and prodrugs thereof; providedthat when R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO₂, then R²³ is notmethyl, unsubstituted phenyl, or unsubstituted furanyl; provided thatwhen R¹ is OH, R⁴, R⁵, and R⁷ are H, and R⁶ is NO_, then R²⁵ is notunsubstituted phenyl or O-tert-butyl, provided that when R¹ is OH, R⁴,R⁵, R⁷ and R³³ are H, R⁶ is NO₂, then R³² is not dimethylamino; andprovided that when R¹ is OH, R⁴, R⁵, R⁷ and R³³ are H, R⁶ is Br, thenR³² is not dimethylamino. 117-172. (canceled)
 173. A compound of 116,wherein the formula of the compound is:

or a pharmaceutically acceptable, salt, prodrug or ester thereof.